Eosin Methylene Blue (EMB) Agar : Principle purpose and colonies characteristics

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Eosin Methylene Blue MLTGEEKS

Eosin Methylene Blue (EMB, also known as “levine emb agar formulation”) is a selective and differential culture medium for gram-negative bacteria. It’s contains dyes that are toxic to gram-positive bacteria making it selective. EMB is a selective and differential medium for coliforms bacteria. It is made up of two dyes, eosin and methylene blue in the ratio of 6:1 respectively.EMB is a differential microbiological culture media, which slightly inhibits the growth of Gram-positive bacteria and provides a color indicator distinguishing between organisms that ferment lactose (e.g., E. coli) and those that do not (e.g., Salmonella, Shigella species).Organisms that ferment lactose display “nucleated colonies“—colonies with dark centers.in medical laboratories,This medium is important by distinguishing pathogenic microbes in a short period of time.It is Selective because it encourages some bacteria to grow while inhibiting others.  Eosin Y and Methylene Blue generally inhibits Gram positive bacteria from growing (a few will grow) and generally allow Gram negative organism to grow (some will not grow).

  • If good growth, generally you have a Gram negative bacteria.  
  • If no growth or poor growth, you most likely have a Gram positive.


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 deamination of proteins. This ensures that the dye is not absorbed. The colonies will be colorless.

On EMB if E. coli is grown 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 this way to EMB. This medium has been specifically designed to discourage the growth of gram positive bacteria.Hence EMB is highly differentiale and can be use as a substitute to MacConkeY Agar.

Also read  Müller-Hinton agar principle and antimicrobial susceptibility testing

Purpose of Culturing on EMB Agar

  • To isolate Gram negative organisms
  • To select  and isolate coliforms
  • To differentiate among the family of Enterobacteriaceae
  • It’s main use is to isolate fecal coliforms and to detect  fecal contamination. Both the Gram negative selection and the detection of coliforms is imperfect, a small percentage of strains do not act as expected.

Composition of EMB Agar

uninoculated EMB Agar
uninoculated EMB Agar

This culture media is made up or composed of the following ingredients

  • Enzymatic Digest of gelatin
  • Lactose: Sugar, helps to differentiate lactose fermenter from non
    lactose fermenter
  • Dipotassium Phosphate
  • Eosin Y: Indicator
  • Methylene Blue: pH indicator
  • Agar

Culturing on EMB Agar

  1. Appropriately label you plates.  Mark the dish bottom into thirds and label what species will be in each section.  It is best to label the side of the bottom plate or write on the bottom in tiny letters so that you will be able to observe the growth clearly.
  2. Inoculate one third with your unknown streak for isolated colonies if possible.  Isolated colonies work best for this test but it is not essential.
  3. Inoculate one third with E. coli, and the other third with S. epidermidis.  Generally it is best to keep E. coli well away from the other cultures because it may overgrow them.  (Streak only one species/third, don’t mix the bacteria!  See later for how to do this quickly.)
  4. You will want to compare the growth on these plates to growth inoculated on a general purpose media such as NA. or TSA.
  5. Incubate your plates upside down in the incubator at 35-37 C for 1-2 days.  If possible, exam plates at both 1 and 2 days.
  6. Slow growing species may require a day or two of additional growth.
Also read  Parasitology Case study #2

Observation on EMB Agar

  1. Compare your growth on these plates to growth on a general purpose media.  (If no growth on the general purpose media, discard your results and repeat the test.)  Look for the presence or absence of growth, and if there is growth if it is reduced from normal.  
  2. If there is growth, check to see the color of the growth.  Is the growth essentially colorless, bright pink, purplish, or greenish with a metallic sheen?  To obtain your results, use the darkest color areas which may be only in the centers of the colonies.
  3. One predicts that if an organism grows well on EMB Agar, it is most likely Gram negative, otherwise it is most likely Gram positive.  And if the growth is good and pink or darker, it is most likely a coliform, otherwise it is likely a noncoliform.  If the colonies are darker than pink (purplish, greenish with a metallic sheen, or even blackish), the organisms highly utilize the sugars lactose/sucrose and have highly lowered the pH.

Differentiation between gram negative bacilli base on colony growth on EMB AGAR

These plates differentiate between species and their ability to lower the pH.

  • If growth is colorless, off-white or a very light and dull pink, the pH has largely been unaffected:  is probably a Gram negative noncoliform.
  • If good growth that is pink, sugar utilization has slightly lowered the pH:  is probably a Gram negative coliform.
  • If good growth that is purplish or has a purple center, sugar utilization has lowered the pH:  is probably a Gram negative coliform.
  • If good growth with a greenish metallic sheen, strong sugar utilization has greatly lowered the pH:  is probably a Gram negative coliform.
  • (If poor or no growth, recall it is probably a Gram positive organism.
Also read  Egg Yolk Agar Base Selective and Differential Medium for the Isolation of Clostridium perfringens

Quality Control of EMB Agar

In order to confirm the tentative conclusions from this culture media,  perform a Gram stain and testing for lactose and sucrose utilization.  Other species of bacteria may be used as positive and negative controls.

  • E. coli will have good purplish or greenish or blackish growth (with or without a metallic sheen),
  • Enterbacter aerogenes will have good pink or purplish growth, 
  • S. epidermidis will grow poorly if at all. 

 And yes, the same species can produce different colors depending on how far the pH shifts. 

EMB agar is only moderately inhibitive to many Gram positives and yeasts while some strains of Salmonella and Shigella may not grow well. 

The greenish metallic sheen does not always appear on strains that produce it.

 Sterilization of this media reduces the methylene blue and creates a precipitate which may be oxidized back and dispersed respectively by mixing the media.

proteus mirabelis and e coli on emb agar
proteus mirabelis and e coli on emb agar

Source http://spot.pcc.edu/

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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