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Eosin-Methylene Blue Agar Plates Protocol Send Print

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Created: Saturday, 29 September 2007
Last update: Monday, 01 April 2013
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History

Eosin-methylene blue (EMB) agar was first developed by Holt-Harris and Teague in 1916.  They used EMB agar to clearly differentiate between the colonies of lactose fermenting and nonfermenting microbes.  In the same medium, sucrose was also included to differentiate between coliforms that were able to ferment sucrose more rapidly than lactose and those that were unable to ferment sucrose.  Lactose fermenter colonies were either black or possessed dark centers with transparent and colorless outer margins, while lactose or sucrose nonfermenters were colorless.  EMB agar was shown to be more sensitive and stable and differentiated between sugar fermenters and nonfermenters faster when compared to other agars.

In 1918, Levine described an EMB agar that differentiated between fecal and nonfecal types of the coli aerogenes group.   It also differentiated between salmonellae and other nonlactose fermenters from the coliforms.

Present day Bacto EMB agar is a combination of the EMB agar described by Holt-Harris and Teague and Levine.  It contains lactose and sucrose (as described by Holt-Harris and Teague) and also contains Bacto peptone and phosphate (as described by Levine).  The two indicator dyes, eosin and methylene blue, are used in a ratio to impart minimum toxicity but provide best differentiation.

Purpose

Eosin-methylene blue agar is selective for gram-negative bacteria against gram-positive bacteria.  In addition, EMB agar is useful in isolation and differentiation of the various gram-negative bacilli and enteric bacilli, generally known as coliforms and fecal coliforms respectively (8).  The bacteria which ferment lactose in the medium form colored colonies, while those that do not ferment lactose appear as colorless colonies (1).  EMB agar is used in water quality tests to distinguish coliforms and fecal coliforms that signal possible pathogenic microorganism contamination in water samples.  EMB agar is also used to differentiate the organisms in the colon-typhoid-dysentery group:  Escherichia coli colonies grow with a metallic sheen with a dark center, Aerobacter aerogenescolonies have a brown center, and nonlactose-fermenting gram-negative bacteria appear pink (5).

Theory

EMB agar contains peptone, lactose, sucrose, and the dyes eosin Y and methylene blue; it is commonly used as both a selective and a differential medium.  EMB agar is selective for gram-negative bacteria.  The dye methylene blue in the medium inhibits the growth of gram-positive bacteria; small amounts of this dye effectively inhibit the growth of most gram-positive bacteria (8). Eosin is a dye that responds to changes in pH, going from colorless to black under acidic conditions.  EMB agar medium contains lactose and sucrose, but not glucose, as energy sources.  The sugars found in the medium are fermentable substrates which encourage growth of some gram-negative bacteria, especially fecal and nonfecal coliforms.  Differentiation of enteric bacteria is possible due to the presence of the sugars lactose and sucrose in the EMB agar and the ability of certain bacteria to ferment lactose in the medium.  Lactose-fermenting gram-negative bacteria (generally enteric) acidify the medium, and under acidic conditions the dyes produce a dark purple complex which is usually associated with a green metallic sheen.  This metallic green sheen is an indicator of vigorous lactose and/or sucrose fermentation ability typical of fecal coliforms.   A smaller amount of acid production, which is a result of slow fermentation (by slow lactose-fermenting organisms), gives a brown-pink coloration of growth.  Colonies of nonlactose fermenters appear as translucent or pink (6, 9).

RECIPE

As described in the Difco manual (2):

10 g of Bacto peptone
5 g of Bacto lactose
5 g of Bacto sucrose
2 g of dipotassium phosphate
13.5 g of Bacto agar
0.4 g of Bacto eosin Y
0.065 g of Bacto methylene blue
Distilled water t o bring final volume to 1 liter.

Adjust pH to 7.2.  Boil to completely dissolve agar.  Sterilize in an autoclave for 15 minutes at 15 psi (121oC).  Cool to 60oC.  If any precipitate is apparent in the medium, disperse by gently swirling before pouring into sterile Petri dishes (1).

EMB agar is commercially available in premixed form from biological supply companies.

PROTOCOL

Obtain an EMB agar plate and streak it with the appropriate bacterial culture using the quadrant streak plate method.  This will result in the isolation of individual colonies.
 


FIG. 1. Nutrient agar inoculated with Escherichia coli (a gram-negative bacterium) demonstrating growth with whitish colonies.

 

FIG. 2. EMB agar inoculated with Escherichia coli (a gram-negative bacterium) demonstrating growth with green-metallic sheen colonies (for details see the Atlas page).



FIG. 3. Nutrient agar inoculated with Pseudomonas aeruginosa (a gram-negative bacterium) demonstrating growth with whitish colonies. 



FIG. 4. EMB agar inoculated with Pseudomonas aeruginosa (a gram-negative bacterium) demonstrating growth with pinkish mucoid colonies (for details see the Atlas page).



FIG. 5. Nutrient agar inoculated with Staphylococcus aureus (a gram-positive bacterium) demonstrating growth with whitish colonies.



FIG. 6. EMB agar inoculated with Staphylococcus aureus (a gram-positive bacterium) demonstrating no growth.  EMB medium inhibits the growth of gram-positive bacteria.

SAFETY

The ASM advocates that students must successfully demonstrate the ability to explain and practice safe laboratory techniques. For more information, read the laboratory safety section of the ASM Curriculum Recommendations: Introductory Course in Microbiology and the Guidelines for Biosafety in Teaching Laboratories.

COMMENTS AND TIPS

The concentration of agar may be increased to 5% (by using an additional 3.65 g of agar per 1 liter of medium, refer to the recipe section) to inhibit the spreading of Proteus (1).

If the sucrose-containing EMB medium is used, Proteus colonies will also show the characteristic metallic sheen if they are inhibited from spreading by the higher concentration of agar (1).


Besides being used as a fermentation indicator medium to differentiate gram-negative enteric bacteria, EMB can also be used for testing strains of bacteria for sensitivity to phage.  In this case, 5 g of NaCl per liter is to be added into the medium, and the medium is to be made without added sugars to a final concentration of 1% as in the typical EMB.  This medium is then designated as EMBO agar (3).

REFERENCES

1.  Bailey, W. R., and E. G. Scott.  1966. Diagnostic microbiology: a text book for the isolation and identification of pathogenic microorganisms, 2nd ed. The C. V. Mosby Company, St. Louis, MO.
2. Difco Laboratories. 1984. Difco manual, 10th ed., p. 324–326.   Difco Laboratories, Inc., Detroit, MI.
3. Gerhardt, P., R. G. E. Murray, W. A. Wood, and N. R. Krieg.  1994. Methods for general and molecular bacteriology.  American Society for Microbiology, Washington, DC.
4. Holt-Harris, J. E., and O. Teague.  1916. A new culture medium for the isolation of Bacillus typhosus from stools. J. Infect. Dis. 18:596–600.
5. Jacobs, M. B., and M. J. Gerstein.  1960. Handbook of microbiology. D. Van Nostrand Company Inc., Princeton, NJ.
6. Leboffe, M. J., and B. E. Pierce. 2005. A photographic atlas for microbiology laboratory, 3rd ed. Morton Publishing, Englewood, CO.
7. Levine, M. 1918. Differentiation of B. coli and B. aerogenes on a simplified eosin-methylene blue agar. J. Infect. Dis. 23:43–47.
8. Madigan, M. T., J. M. Martinko, and J. Parker.  2006. Brock biology of microorganisms: an international edition, 11th ed. Prentice-Hall International, Inc., Upper Saddle River, NJ.
9. Prescott, L. M., J. P. Harley, and D. A. Klein.  1993. Microbiology, 2nd ed. Wm. C. Brown Communication, Inc., Boston, MA.

REVIEWERS

This resource was peer-reviewed at the ASM Conference for Undergraduate Educators 2007.

Participating reviewers:

Joel Adams-Stryker
Evergreen Valley College, San Jose, CA

Robert Bauman
Amarillo College, Amarillo, TX

Dexter Beck
Chattahoochee Technical College, Marietta, GA

Donald Breakwell
Brigham Young University, Provo, UT

Elaine Bukowski
Ivy Tech Community College of Indiana, Prospect, KY

Cheryl Dias
Harold Washington College, Chicago, IL

Edith Hillard
Frederick Community College, Frederick, MD

Catherine A. Hopper
University of Maine, Orono, ME

Joanna Klein
Northwestern College, St. Paul, MN

Alexandra Kurtz
Georgia Gwinnett College, Lawrenceville, GA

Sue Merkel

Cornell University, Ithaca, NY

Heidi R. Smith
Front Range Community College, Fort Collins, CO

Sherry Stewart
Navarro College, Corsicana, TX 
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