EMB Agar: Composition, Principle, and Colony Morphology

Eosin Methylene Blue (EMB) agar is a selective and differential culture medium. It selectively promotes the growth of Gram-negative bacteria and aids in the differentiation of lactose fermenter and non-lactose fermenting colonies.

EMB Agar is used for the isolation of fecal coliforms. EMB Agar can be streaked for isolation or used in the Membrane Filter Technique. EMB agar inoculated with Escherichia coli demonstrates growth with green-metallic sheen colonies.

EMB agar, first described by Holt-Harris and Teague, contained lactose and sucrose as sources of carbohydrates. Levine modified the medium by adding peptone and phosphate, removing sucrose from the formula, and increasing the lactose content. This aided in differentiating fecal and non-fecal types of coliforms. It also differentiates salmonellae and other non-lactose fermenters from the coliforms.

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. 

Principle

EMB agar contains sucrose and lactose, utilized as fermentable carbohydrates substrates, which encourage the growth of some gram-negative bacteria, especially fecal and non-fecal 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 the lactose in the medium.

• Lactose-fermenting gram-negative bacteria acidify the medium, which reduces the pH, and the dye produces a dark purple complex usually associated with a green metallic sheen. This metallic green sheen indicates vigorous lactose and/or sucrose fermentation ability typical of fecal coliforms.
• Organisms that are slow lactose-fermenters produce less acid, and the colonies appear brown-pink.
• Non-lactose fermenters increase the pH of the medium by deamination of proteins and produce colorless or light pink colonies.

Eosin Y and methylene blue are pH indicator dyes that combine to form a dark purple precipitate at low pH; they also inhibit the growth of most Gram-positive organisms. Peptic digest of animal tissue is a source of carbon, nitrogen, and other essential growth nutrients. Phosphate buffers the medium.

Composition of EMB Agar

The composition of EMB agar and modified EMB agar (Levine EMB) agar differs slightly. Levine modification contains 10g of lactose (twice as in EMB agar) and no sucrose.

Ingredients	EMB agar (gm/L)	Levine EMB agar (gm/L)
Peptone	10 g	10g
Lactose	5 g	10g
Sucrose	5g	–
Dipottasium,PO4	2g	2g
Agar	13.5g	13.5g
Eosin Y	0.4g	0.4g
Methylene blue	0.065g	0.065g

Preparation of EMB agar

1. Weigh and suspend 35.96 grams of dehydrated media in 1000 ml distilled water.
2. Mix until the suspension is uniform and heat to boiling to dissolve the medium completely.
3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. 
4. Cool to 45-50°C, and with frequent gentle swirling, pour the media into sterile Petri plates. Note: frequent swirling is recommended to restore the blue color o methylene blue and to suspend the flocculent precipitate, if any.
5. Label with initials of the name of the medium, and the date of preparation.
6. Store the plates upside down (lids below) in the refrigerator until use.

Colony Morphology

Organism	Colonial appearance on EMB agar
Escherichia coli	Colonies are 2-3 mm in diameter and have a greenish metallic sheen in reflected light, dark or even black center in transmitted light.
Enterobacter aerogenes	Colonies are 4-6mm in diameter, raised and mucoid, tending to become confluent. No metallic sheen, grey-brown centers by transmitted light
Salmonella and Shigella spp	Translucent and colorless colonies
Pseudomonas spp	Colorless irregular colonies
Proteus spp	Colorless colonies
Gram-positive cocci	Partially inhibited or no growth
Coagulase-positive staphylococci	Colorless, “pin-point” colonies on modified EMB

Quality Control of EMB agar

Sterility testing can be performed by incubating 3-5% uninoculated plates from each batch at 37°C for 18-24 hours. Any growth in the media should be regarded as contamination, and the whole lot should be discarded.

Performance testing of prepared EMB agar plates can be done by inoculating known strains of bacteria into the medium and observing growth and colonial characteristics.

Organism	Growth and colony characteristics
E.coli ATCC 25922	Good growth, blue-black colonies with a green metallic sheen
Salmonella choleraesuis subsp. Choleraesuis serotype Typhimurium ATCC 14028	Luxuriant growth, colorless to amber colonies
Enterococcus faecalis ATCC 29212	Inhibition (partial)
Shigella flexneri ATCC 12022	Moderate to heavy growth, colorless to amber colonies

Uses of EMB agar

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 water sample indicates the possibility of fecal contamination of water, so does the presence of other pathogenic enterics).
2. EMB media assists in the visual distinction of Escherichia coli, other nonpathogenic lactose-fermenting enteric gram-negative rods, and the Salmonella and Shigella genera. Escherichia coli colonies grow with a metallic sheen with a dark center. Aerobacter aerogenes colonies have a brown center, and non-lactose-fermenting gram-negative bacteria appear pink.
3. 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.
4. Levine EMB Agar can be used to isolate and identify Candida albicans from clinical specimens. Addition of 0.1g/L of chlortetracycline hydrochloride after autoclaving makes the medium selective by inhibiting the accompanying bacterial flora. The culture medium then is blue in color. Colonies of Candida albicans appear `spidery’ or `feathery’ after 24 to 48 hours of incubation at 35°C in 10% carbon dioxide. Other Candida species produce smooth yeast-like colonies.

References and further readings

• ASMscience | Eosin-Methylene Blue Agar Plates Protocol. Retrieved June 12, 2020, from https://www.asmscience.org/content/education/protocol/protocol.2869
• Welcome to Microbugz—Eosin Methylene Blue Agar. Retrieved June 12, 2020, from https://www.austincc.edu/microbugz/eosin_methylene_blue_agar.php
• Eosin Methylene Blue Agar (EMB). (2016, April 12). Biology LibreTexts.