EMB Agar- Composition, Principle, Preparation, Results, Uses

Eosin Methylene Blue (EMB) agar is a selective and differential medium that is used for the isolation and differentiation of gram-negative enteric bacteria from clinical and non-clinical specimens. It was initially formulated in 1916 by Holt-Harris and Teague, who used eosin and methylene blue dyes to visibly distinguish between lactose-fermenting and non-lactose-fermenting microorganisms. Later, Levine modified the medium by adding peptone and phosphate, removing sucrose, and increasing the lactose content. This improved the differentiation of fecal and non-fecal types of coliforms.

EMB agar is commonly used in clinical microbiology to detect the presence of specific microbes associated with disease or poor sanitation, such as Escherichia coli, Salmonella, Shigella, and other enteric pathogens. It is also used in testing the quality of water, especially in determining if the water is contaminated by harmful microorganisms.

EMB agar is a complex medium that contains the following ingredients:

• Peptic digest of animal tissue: This provides the essential nutrients, such as carbon, nitrogen, vitamins, and minerals, for the growth of bacteria.
• Lactose and sucrose: These are fermentable carbohydrates that serve as energy sources for bacteria. They also help to differentiate lactose-fermenting and non-lactose-fermenting bacteria based on the acid production and dye uptake.
• Eosin Y and methylene blue: These are synthetic dyes that act as pH indicators and selective agents. They inhibit the growth of gram-positive bacteria and some gram-negative bacteria. They also impart different colors to the colonies of bacteria depending on their ability to ferment lactose and/or sucrose and produce acid.
• Dipotassium phosphate: This acts as a buffer to maintain the pH of the medium around 7.2.
• Agar: This is a solidifying agent that provides a firm surface for bacterial growth.

The final pH of EMB agar is 7.2±0.2 at 25°C. The ratio of eosin Y and methylene blue is 6:1.

EMB agar is characterized by the presence of a combination of the two dyes eosin and methylene blue in the ratio of 6:1. These dyes act as both selective and differential agents in the medium. They inhibit the growth of most gram-positive bacteria and some gram-negative bacteria, while allowing the growth of enteric bacteria. They also react with the acidic products of lactose fermentation and produce distinctive color changes in the colonies.

Gram-negative bacteria that ferment lactose produce acid which lowers the pH of the medium. This encourages dye absorption by the colonies and turns them dark purple as the acid acts upon the dyes. In addition, certain lactose-fermenting bacteria produce flat, dark colonies with a green metallic sheen. This is due to rapid fermentation of lactose and production of strong acids, which reduce the methylene blue to a colorless compound and form a precipitate with eosin. The most common example of this type of bacteria is Escherichia coli.

Other lactose-fermenting bacteria produce larger, mucoid colonies, often purple only in their center. These bacteria produce less acid and do not reduce methylene blue completely. An example of this type of bacteria is Enterobacter aerogenes.

Lactose non-fermenting bacteria, on the other hand, do not produce acid and may increase the pH by deamination of proteins. This ensures that the dye is not absorbed by the colonies and they remain colorless or light lavender. These bacteria also do not reduce methylene blue and do not form a precipitate with eosin. Examples of this type of bacteria are Salmonella and Shigella.

Peptic digest of animal tissue serves as a source of carbon, nitrogen, and other essential growth nutrients for the bacteria. Lactose and sucrose are the sources of energy by being fermentable carbohydrates. Phosphate buffers the medium and prevents drastic changes in pH.

EMB agar is thus a useful medium for isolating and differentiating gram-negative enteric bacteria based on their ability to ferment lactose and their reaction with the dyes.

EMB agar is a solid medium that can be prepared from a dehydrated powder or from scratch using the following ingredients:

• Peptic digest of animal tissue: 10 g
• Lactose: 5 g
• Sucrose: 5 g
• Dipotassium phosphate: 2 g
• Eosin-Y: 0.4 g
• Methylene blue: 0.065 g
• Agar: 13.5 g
• Distilled water: 1000 ml

The steps for preparing and using EMB agar are as follows:

1. Suspend the ingredients in distilled water and mix well until the suspension is uniform.
2. Heat the mixture to boiling to dissolve the medium completely. Avoid overheating as it may affect the pH and the dyes.
3. Sterilize the medium by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
4. Cool the medium to 45-50°C and shake it gently to oxidize the methylene blue (i.e. to restore its blue color) and to suspend the flocculent precipitate.
5. Pour the medium into sterile Petri plates and let them solidify.
6. Allow the plates to warm to room temperature before inoculating. The agar surface should be dry and free of condensation.
7. Inoculate and streak the specimen as soon as possible after collection using a sterile loop or swab. If the specimen is on a swab, roll it over a small area of the agar surface and then streak for isolation with a loop.
8. Incubate the plates aerobically at 35-37°C for 18-24 hours and protect them from light.
9. Examine the plates for colonial morphology and color. If negative after 24 hours, reincubate for an additional 24 hours.

The results on EMB agar depend on the ability of the bacteria to ferment lactose and/or sucrose and the production of acid. The dyes eosin and methylene blue act as pH indicators and change color according to the acidity of the medium. The following table summarizes the possible results on EMB agar:

Gram-positive bacteria are inhibited by the dyes in the medium and show no growth or poor growth. Gram-negative bacteria can grow on the medium and show different colony colors depending on their fermentation ability. Non-fermenters do not produce acid and do not change the color of the dyes, resulting in colorless or light lavender colonies. Slow fermenters produce weak acid and change the color of the dyes slightly, resulting in pink or purple colonies. Strong fermenters produce strong acid and change the color of the dyes dramatically, resulting in dark purple colonies with a green metallic sheen. The green metallic sheen is caused by the precipitation of the dyes on the surface of the colonies.

The most characteristic result on EMB agar is the green metallic sheen of E. coli colonies, which indicates a rapid and vigorous fermentation of lactose and/or sucrose and a strong acid production. However, not all strains of E. coli produce this sheen, and some other lactose-fermenting bacteria may also produce it. Therefore, the green metallic sheen is not diagnostic for E. coli and additional tests are needed to confirm its identity.

EMB agar is useful for isolating and differentiating gram-negative enteric bacteria from clinical and non-clinical specimens, especially for detecting coliforms, which are lactose-fermenting bacteria that indicate fecal contamination. However, EMB agar has some limitations, such as:

• A non-selective medium should be inoculated in conjunction with EMB agar to ensure the recovery of all bacteria present in the specimen.
• Some strains of Salmonella and Shigella may fail to grow or grow poorly on EMB agar.
• Some gram-positive bacteria, such as enterococci, staphylococci, and yeasts may grow on EMB agar and form pinpoint colonies.
• Non-pathogenic, non-lactose-fermenting organisms may also grow on EMB agar and be confused with pathogenic strains.
• Serial inoculation may be required to ensure adequate isolation of mixed flora samples.

Therefore, EMB agar should be used in combination with other biochemical, immunological, molecular, or mass spectrometry tests for complete identification of bacteria.

EMB agar is a selective and differential medium that is widely used for the isolation and differentiation of gram-negative enteric bacteria from clinical and non-clinical specimens. Some of the common uses of EMB agar are:

• It is useful in differentiating gram-positive and gram-negative bacteria based on their ability to grow on the medium. Gram-positive bacteria are inhibited by the dyes eosin and methylene blue, while gram-negative bacteria can grow on the medium.
• It helps in the isolation and differentiation of enteric bacilli and gram-negative bacilli based on their fermentation of lactose and sucrose. Lactose-fermenting bacteria produce acid that lowers the pH and causes the colonies to absorb the dyes and turn dark purple or black. Some lactose-fermenting bacteria, such as E. coli, produce a characteristic green metallic sheen on the medium due to rapid fermentation of lactose and production of strong acids. Non-lactose-fermenting bacteria do not produce acid and remain colorless or light lavender on the medium.
• It is used in testing the quality of water, especially in determining if the water is contaminated by harmful microorganisms such as fecal coliforms. EMB agar can detect the presence of coliforms by their lactose fermentation and green metallic sheen. The presence of coliforms indicates fecal contamination and possible presence of other pathogens in the water.
• It differentiates microorganisms in the colon-typhoid-dysentery group based on their morphology and color on the medium. For example, E. coli produces green metallic sheen colonies, Enterobacter produces dark purple colonies with a dark center, Klebsiella produces mucoid purple colonies, Salmonella produces colorless colonies with black centers, Shigella produces colorless colonies, and Proteus produces colorless colonies with a brown halo.
• It also helps in the isolation and differentiation of lactose fermenting and non-lactose fermenting enteric bacilli based on their ability to produce gas from lactose fermentation. Gas production causes cracks or bubbles in the agar around the colonies. For example, E. coli produces gas from lactose fermentation, while Shigella does not.

EMB agar is a useful medium for screening and identifying gram-negative enteric bacteria from various sources. However, it has some limitations that require further confirmation by biochemical, immunological, molecular, or mass spectrometry testing for complete identification.

• EMB Agar is not a definitive medium for the identification of gram-negative enteric bacteria. It only provides preliminary information based on the colonial morphology and color. A non-selective medium should be inoculated in conjunction with EMB Agar to ensure the growth of all organisms present in the specimen. It is also recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
• EMB Agar may not support the growth of some strains of Salmonella and Shigella, which are important pathogens in the colon-typhoid-dysentery group. These organisms may be inhibited by the high concentration of dyes in the medium or may produce atypical colonies that are difficult to recognize. Therefore, other selective and differential media, such as XLD Agar or SS Agar, should be used along with EMB Agar for the isolation and differentiation of Salmonella and Shigella.
• EMB Agar may not differentiate some gram-positive bacteria, such as enterococci, staphylococci, and yeast, from gram-negative bacteria. These organisms may grow on this medium and usually form pinpoint colonies that are colorless or light lavender. However, they can be easily distinguished from gram-negative bacteria by performing a Gram stain or other simple tests, such as catalase or coagulase.
• EMB Agar may not differentiate some non-pathogenic, non-lactose-fermenting organisms from pathogenic strains. For example, Proteus, Morganella, and Providencia species may grow on this medium and produce colorless colonies that resemble those of Salmonella or Shigella. However, these organisms can be differentiated from Salmonella or Shigella by performing additional biochemical tests, such as indole, urease, or citrate.
• EMB Agar may not produce a characteristic green metallic sheen for some strains of E. coli. The green metallic sheen is caused by the rapid fermentation of lactose and production of strong acids that lower the pH and act upon the dyes. However, some strains of E. coli may ferment lactose slowly or weakly and produce less acid or more gas. These strains may produce larger, mucoid colonies that are purple only in their center or have no sheen at all. Therefore, the green metallic sheen is not diagnostic for E. coli and other tests should be performed to confirm its identity.

Source: https://microbeonline.com/eosin-methylene-blue-emb-agar-composition-principle-uses-preparation-and-result-interpretation/

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