Bile Esculin Agar- Composition, Principle, Preparation, Results, Uses

Bile Esculin Agar (BEA) is a type of culture medium that is used in microbiology to isolate and identify certain bacteria, especially those belonging to the genus Enterococcus. Enterococci are gram-positive cocci that are commonly found in the gastrointestinal tract of humans and animals, and can cause various infections such as urinary tract infections, endocarditis, and bacteremia. Enterococci were formerly classified as group D streptococci, but they were reclassified in their own genus in 1984 based on molecular and biochemical criteria.

Bile esculin agar (BEA) is a selective and differential medium that contains the following ingredients per liter of distilled water:

• Agar: 15 g
• Esculin: 1 g
• Ferric citrate: 0.5 g
• Beef extract: 11 g
• Enzymatic digest of gelatin: 34.5 g
• Ox bile: 40 g

The final pH of the medium is 6.6±0.2 at 25°C.

Bile Esculin Agar (BEA) is a selective and differential medium that exploits the ability of certain bacteria, especially enterococci and group D streptococci, to grow in the presence of bile and hydrolyze esculin.

Bile Esculin Agar (BEA) is a solid medium that can be prepared by following these steps:

1. Suspend 64.5 grams of BEA powder in 1000 ml of distilled water.
2. Heat to boiling with agitation to dissolve the medium completely.
3. Dispense into tubes or flasks as desired.
4. Sterilize by autoclaving at 121°C for 15 minutes.
5. Allow the tubed medium to solidify in a slanted position with a butt of 2.5 cm deep or pour into sterile Petri plates.
6. Allow the BEA medium to cool to room temperature before use.

The result of the bile esculin test is based on the ability of the organism to hydrolyze esculin in the presence of bile. Esculin hydrolysis produces esculetin, which reacts with ferric ions in the medium to form a dark brown to black complex. This color change indicates a positive result for esculin hydrolysis.

Bile Esculin Agar (BEA) is a useful medium for several purposes:

• It is recommended for use as a differential medium in the isolation and presumptive identification of enterococci/group D streptococci. It can also be used to differentiate these organisms from viridans streptococci and other gram-positive microorganisms.
• It is used in conjunction with other biochemical tests to identify cultures of isolated organism. BEA is used for the presumptive identification of Enterobacter, Klebsiella spp., and Serratia spp., among the Enterobacteriaceae.
• The use of the bile esculin test on this medium is a reliable way of identifying group D streptococci from non-group D streptococci.
• Bile Esculin Agar is recommended for the isolation and identification of Yersinia enterocolitica from food and animal feeding stuffs.

Bile Esculin Agar is a useful medium for the presumptive identification of group D streptococci and enterococci, but it has some limitations that should be considered:

• The medium is not suitable for the primary isolation of patient specimens, as it may inhibit the growth of some clinically relevant organisms. It should be used only with pure cultures of isolated organisms.
• The medium is not specific for group D streptococci and enterococci, as some other gram-positive and gram-negative bacteria may also grow and hydrolyze esculin on this medium. For example, some strains of Staphylococcus, Aerococcus, Listeria monocytogenes, Enterobacter, Klebsiella, Serratia, and Yersinia enterocolitica may produce black colonies on Bile Esculin Agar. Therefore, additional tests are required to confirm the identity of the isolates.
• The medium may give false-negative results for some group D streptococci and enterococci that do not hydrolyze esculin or grow poorly in the presence of bile. For example, some strains of Streptococcus bovis and Enterococcus gallinarum may not produce black colonies on Bile Esculin Agar. Therefore, negative results should be verified by other methods.
• The medium may give false-positive results for some organisms that hydrolyze esculin but are not group D streptococci or enterococci. For example, some strains of Streptococcus pyogenes and Streptococcus agalactiae may produce black colonies on Bile Esculin Agar. Therefore, positive results should be verified by other methods.
• The medium may be difficult to interpret if the inoculum is too heavy or if there is contamination by other organisms. A heavy inoculum may reduce the inhibitory effect of bile and allow the growth of non-group D streptococci or non-enterococci that may hydrolyze esculin. Contamination by other organisms may obscure the color change of the medium or produce mixed reactions. Therefore, care should be taken to inoculate the medium with a single isolated colony and to avoid cross-contamination.

In summary, Bile Esculin Agar is a convenient and rapid screening test for group D streptococci and enterococci, but it should not be used as the sole criterion for identification. Other biochemical, immunological, molecular, or mass spectrometry tests should be performed on colonies from pure culture for complete identification. Bile Esculin Agar should also be used in conjunction with other selective and differential media to isolate and identify other clinically relevant organisms.

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