Eosin Methylene Blue Agar

Eosin Methylene Blue (or EMB) Agar is a Selective & Differential Medium.  The selective and differential aspects are due to the dyes Eosin Y and Methylene Blue, and the sugars lactose and sucrose in the medium.  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).

The medium is Differential because of the dyes Eosin Y and Methylene Blue, and the sugars lactose and sucrose.  The dyes turn darker as the pH lowers, starting with colorless off-white colonies (or a very light and dull pink), to pink, to purple, to dark green with a metalic sheen.  This darkening may only be in the colony center and may sometimes be so dark that it appears black.  If either of the two sugars is utilized, the acid products will reduce the pH.  Strong lactose and sucrose utilization will lower the pH further than weak utilization.  These plates differentiate between species and their ability to lower the pH.

Purpose:  to select for and isolate Gram negative organisms, to select for and isolate coliforms, and to differentiate among the family of Enterobacteriaceae.  Its main use is to isolate fecal coliforms and to detect for fecal contamination.  Both the Gram negative selection and the detection of coliforms is imperfect, a small percentage of strains do not act as expected.

Procedure:  

  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.
Results  
  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.

References:

Difco Laboratories. 1998. Difco Manual, 11th ed. Difco Laboratories, Sparks, MD, USA.

Harley J.P. and Prescott L.M. 2002. Laboratory Exercises in Microbiology, 5th ed. McGraw-Hill Higher Education, New York, NY, USA.

MacFaddin J.F. 2000. Biochemical Tests for the Identification of Medical Bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA, USA.

Instructor Notes:

Students should confirm their tentative conclusions from this test by performing 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, and S. epidermidis will grow poorly if at all.  And yes, the same species can produce different colors depending on how far the pH shifts.  Instructors may wish to have students test several selective and differential media at the same time and then compare them all to growth on one general purpose media plate to save on the cost.  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.