EMB Agar: Composition, Principle, and Colony Morphology

A water quality inspector sends a sample from a municipal tap to the laboratory, concerned about fecal contamination following recent flooding. The technician plates the sample onto EMB agar. Within 24 hours, colonies with a striking metallic green sheen appear under reflected light: the unmistakable signature of Escherichia coli, a confirmed indicator of fecal contamination.

That metallic green sheen is one of the most distinctive colony appearances in all of bacteriology, and it is produced only by organisms that ferment lactose so vigorously that the acidified medium causes eosin and methylene blue dyes to combine into a dark precipitate that reflects green light.

EMB agar is used wherever you need to (1) select for Gram-negative enteric organisms, and (2) immediately distinguish the vigorous lactose fermenters (the fecal coliforms) from everything else.

Introduction

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.

Figure: Escherichia coli colonies in Eosin Methylene Blue Agar (Note: Greenish Metallic Sheen)

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.

EMB agar serves two simultaneous purposes: selection and differentiation, making it one of the most informative primary plating media in clinical microbiology:

• Selective: Eosin Y and methylene blue dyes inhibit gram-positive organisms and most fastidious gram-negative bacteria, allowing gram-negative enteric bacilli to grow
• Differential: Lactose as the fermentable sugar, with eosin and methylene blue as pH-sensitive dyes, produces dramatically different colony appearances depending on how vigorously an organism ferments it, ranging from the metallic green sheen of vigorous fermenters to the colorless colonies of non-fermenters

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 lactose as the fermentable carbohydrate. The original Holt-Harris and Teague formulation also contained sucrose, but the Levine modification used in most laboratories today removes sucrose and doubles the lactose. Differentiation depends on how vigorously an organism ferments the available sugar and therefore how much acid it produces at the colony surface.

• 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 the vigorous lactose fermentation 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.

Why E. coli produces metallic green sheen

The characteristic metallic green sheen of E. coli on EMB agar is one of the most recognizable and diagnostically significant colony appearances in microbiology. Understanding why it occurs requires understanding the chemistry of the dye complex:

E. coli is a vigorous fermenter of lactose. This fermentation produces so much acid that the pH drops dramatically at the colony surface. At this very low pH:

1. Eosin Y and methylene blue precipitate out of solution and are absorbed in large quantities by the colony
2. The massive dye complex formed at very low pH produces a dark purple-black colony color
3. The surface of the colony develops a metallic iridescence due to light interference from the densely packed dye crystals, similar to the iridescent sheen seen on a soap bubble or oil film on water

This metallic green sheen is therefore a direct indicator of vigorous acid production from lactose fermentation, a hallmark of E. coli among the Enterobacteriaceae. On the original sucrose-containing formulation, sucrose fermenters could also produce a sheen, which is precisely why Levine removed it. Organisms that ferment lactose less vigorously (like Klebsiella) may produce dark colonies but without the metallic sheen.

Composition of EMB Agar

The original EMB agar (Holt-Harris and Teague) contained both lactose and sucrose. Levine modified the formula by removing sucrose and doubling the lactose content: this modification reduces the number of false-positive metallic sheen colonies from sucrose-only fermenters and improves discrimination of true fecal coliforms. Levine EMB is the formulation used in most clinical and water microbiology laboratories today.

pH: 7.1 ± 0.2 at 25 °C.

Why two dyes? Eosin Y alone would give a reddish precipitate. Methylene blue alone would give a blue one. Together, at acidic pH, they form a dark purple-black complex that has the unique property of creating a metallic green sheen when the precipitate is dense enough (as in vigorous fermentation). The combination is synergistic: neither dye alone produces the metallic sheen.

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 of 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.

Inoculation and Incubation

1. Allow the plate to reach room temperature and dry the agar surface before use.
2. Inoculate directly from the specimen. Suitable specimens include urine, stool, wound swabs, and, in water microbiology, the membrane filter after filtering a measured water volume.
3. If culturing from a swab, roll the swab over a small area at the edge of the plate first, then streak out for isolation with a sterile loop.
4. Incubate aerobically at 35 to 37°C. EMB does not require carbon dioxide.
5. Read at 18 to 24 hours for the primary result, and again at 48 hours. Late lactose fermenters such as Citrobacter freundii and Shigella sonnei can appear colorless or pale at 24 hours and darken by 48 hours, so an early-only read can misclassify a fermenter as a non-fermenter.
6. For the membrane filter technique in water testing, place the filter on the EMB surface and count metallic-sheen colonies at 24 to 48 hours as presumptive fecal coliforms.

Reading on EMB plate

Figure: EMB agar inoculated with Escherichia coli demonstrating growth with green-metallic sheen colonies. (Image source: Carmen Moreno González)

Examine the plate in reflected light (room light shining down onto the plate surface) for metallic sheen. Then hold the plate up to a transmitted light source (light shining through from below) to see the dark centers. Missing the reflected light step is the most common cause of misread EMB plates: colonies that show metallic green sheen only in reflected light may appear simply as dark colonies in transmitted light.

Quality Control of EMB agar

Sterility testing can be performed by incubating 3 to 5 uninoculated plates from each batch at 35 to 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.

Colony Morphology on EMB Agar

Strong lactose fermenters (dark colonies with metallic sheen)

Moderate lactose fermenters (dark pink to brown-pink colonies, no sheen)

Non-lactose fermenters (colorless or transparent colonies)

Organisms inhibited: no growth on EMB agar

Uses of EMB agar

1. Isolation and differentiation of Gram-negative enteric organisms EMB is used as a primary plating medium for clinical specimens where Gram-negative enteric organisms are expected: urine, stool, and wound specimens. It selects for Gram-negatives while simultaneously differentiating lactose fermenters (including E. coli and Klebsiella) from non-fermenters (Salmonella, Shigella).

2. Water quality testing (fecal coliform detection) EMB agar is the standard confirmatory medium for detecting fecal coliforms in drinking water, sewage, and food samples. Samples that produce metallic green sheen colonies on EMB after positive presumptive MPN (Most Probable Number) tests confirm the presence of E. coli, a definitive fecal contamination indicator.

3. Differentiation of E. coli from other coliforms Among lactose-fermenting Gram-negatives, the metallic green sheen specifically identifies E. coli (vigorous fermenter) from Enterobacter/Klebsiella (moderate fermenter, mucoid, no sheen). This distinction is important because E. coli in urine is always clinically significant, while Enterobacter may represent colonization rather than infection.

4. Colon-typhoid-dysentery group differentiation EMB is used alongside MacConkey agar for selective isolation of Salmonella and Shigella from stool specimens. The colorless colonies of these non-fermenters on EMB are picked for confirmatory TSI and serology.

5. Candida isolation (modified Levine EMB) Levine EMB with chlortetracycline (0.1 g/L added after autoclaving) suppresses bacterial flora and supports Candida albicans isolation, which produces characteristic "spidery" or "feathery" colonies under CO₂ conditions.

EMB Agar vs MacConkey Agar

Both EMB and MacConkey are selective and differential media for gram-negative enteric organisms, and they are frequently used together in a primary plating battery. Understanding their differences helps choose the right medium for each application:

The key practical difference: MacConkey is more commonly used in clinical laboratories for routine stool and urine cultures because its neutral red indicator is more stable and the pink/colorless distinction is simpler to read.

EMB is essential in water microbiology where the metallic green sheen provides definitive identification of E. coli as a fecal indicator, and in settings where distinguishing E. coli from Klebsiella colony morphology is required.

When to use EMB over MacConkey:

• When the metallic green sheen of E. coli is specifically needed for presumptive fecal coliform identification (water testing, food microbiology)

When to use MacConkey over EMB:

• Routine clinical specimen plating: MacConkey is less inhibitory for some fastidious gram-negatives
• When observing swarming pattern of Proteus: bile salts in MacConkey better inhibit swarming

In clinical practice: Both are often inoculated simultaneously from the same specimen to maximize information: MacConkey for Enterobacteriaceae identification and Proteus assessment; EMB for presumptive E. coli confirmation by metallic sheen.

→ MacConkey Agar: Composition, Preparation, Uses and Colony Characteristics

How to Remember

EMB = dye-based selection and differentiation

Both eosin Y and methylene blue are dyes that:

• Inhibit Gram-positives (dye uptake disrupts cell membrane function)
• Act as pH indicators (combine at acid pH → dark purple precipitate → metallic green sheen when dense)

The sheen is physics, not chemistry. Remind yourself: the metallic green is light reflecting off a dense dark layer, not a green pigment. This is why it only appears in reflected light and disappears under transmitted light.

Three colony types, three clinical meanings:

EMB vs MacConkey: EMB is for when you need to identify E. coli specifically (water testing, fecal coliform confirmation). MacConkey is for when you need a general enteric screen. In clinical practice, use MacConkey; in water microbiology, use EMB.

Where Students Get Confused

• The green sheen is physics, not a green pigment. The metallic green is light reflecting off a dense layer of precipitated dye, like the sheen on a soap bubble or oil film. It appears only in reflected light (light shining down onto the plate) and can vanish under transmitted light, where the same colony looks simply dark. Reading only in transmitted light is the most common way the sheen is missed.
• Dark colony does not always mean sheen-positive. Klebsiella and Enterobacter ferment lactose enough to make dark pink-brown colonies, but not vigorously enough for the dense dye layer that produces the sheen. Dark and mucoid without sheen points to Klebsiella; dark with a metallic green sheen points to E. coli.
• Colorless does not distinguish Salmonella from Shigella. Both are non-fermenters and both are colorless on EMB. EMB narrows them to the non-fermenter group but cannot separate them. Pick colorless colonies for TSI and serology.
• Late fermenters can read colorless at 24 hours. Citrobacter freundii and Shigella sonnei may look pale at 24 hours and darken by 48 hours. Confirm a non-fermenter appearance with a 48-hour read before reporting it.
• A sheen on the original EMB is not the same as on Levine EMB. On the older sucrose-containing formulation, a sucrose fermenter could also produce a sheen, which is a false positive for E. coli. Levine EMB (lactose only) is used precisely to avoid this. Know which formulation your laboratory uses.

References and further readings

• Tille PM. Bailey & Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.
• Leber AL, editor. Clinical Microbiology Procedures Handbook. 4th ed. Washington, DC: ASM Press; 2016.
• Procop GW, Koneman EW. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th ed. Wolters Kluwer; 2017.
• US Food and Drug Administration. Bacteriological Analytical Manual (BAM). FDA; 2023.
• Holt-Harris JE, Teague O. A new culture medium for the isolation of Bacillus typhosus from stools. J Infect Dis. 1916;18(5):596–600.
• Cheesbrough M. District Laboratory Practice in Tropical Countries, Part 2. 2nd ed. Cambridge University Press; 2006.