Bile Esculin Test- Principle, procedure, results, uses, limitations

The bile esculin test is a biochemical test that is used to identify and differentiate certain bacteria based on their ability to hydrolyze esculin in the presence of bile. Esculin is a compound that consists of glucose and hydroxycoumarin, which are linked by an ester bond. Hydroxycoumarin is a fluorescent molecule that can be detected by its loss of fluorescence when it is broken down. Bile is a digestive fluid that contains bile salts, which inhibit the growth of most Gram-positive bacteria except for some streptococci and enterococci.

The bile esculin test is a biochemical test that aims to identify and differentiate certain groups of bacteria based on their ability to hydrolyze esculin in the presence of bile. The main objectives of the bile esculin test are:

• To identify Enterococci and Group D Streptococci, which are Gram-positive cocci that can grow in a medium with 4% bile salts and hydrolyze esculin to produce a dark brown or black color.
• To differentiate Enterococci and Group D Streptococci from other viridans or non-Group D Streptococci, which are Gram-positive cocci that either cannot grow in a medium with 4% bile salts or cannot hydrolyze esculin.
• To identify Listeria, which are Gram-positive rods that can also grow in a medium with 4% bile salts and hydrolyze esculin.
• To identify some Gram-negative rods, such as Aeromonas and Yersinia enterocolitica, that can hydrolyze esculin without bile.

The principle of the bile esculin test is based on the ability of some bacteria to hydrolyze esculin, a glycosidic compound, in the presence of bile salts. Esculin is composed of glucose and hydroxycoumarin linked by an ester bond. The enzyme esculinase breaks down this bond and releases glucose and esculetin. Esculetin then reacts with ferric citrate, an iron salt present in the medium, to form a dark brown or black complex. This color change indicates a positive result for esculin hydrolysis.

However, not all bacteria that can hydrolyze esculin can do so in the presence of bile salts. Bile salts are inhibitory to most Gram-positive bacteria, except for Enterococci and Group D Streptococci. Therefore, the bile esculin test also selects for bacteria that can grow in a medium with 4% bile salts or 40% bile. This makes the test more specific for Enterococcus and Group D Streptococcus identification.

Alternatively, esculin is a fluorescent compound, and its hydrolysis can be observed by the loss of fluorescence under UV light. This method does not require the addition of ferric citrate to the medium, but it still requires bile salts for selectivity.

The bile esculin test can be performed either on a slant agar medium or on a filter paper disk impregnated with esculin. The disk method is faster and more convenient than the agar method, but it may give false-positive results with some bacteria that produce hydrogen sulfide, which also reacts with iron to form a black precipitate.

The bile esculin test is a simple and reliable test for presumptive identification of Enterococci and Group D Streptococci from other Gram-positive cocci. However, it should be used in conjunction with other biochemical tests to confirm the identity of the isolates.

The bile esculin test is mainly used to test Gram-positive cocci in chains that are catalase-negative and can be morphologically identified as presumed S. bovis. S. bovis is a group of streptococci that includes S. gallolyticus, S. infantarius, and S. lutetiensis. These bacteria are associated with endocarditis and colorectal cancer.

The test can also be used to differentiate enterococci from other pyrrolidonyl-β-naphthylamide (PYR)-positive organisms. Enterococci are a group of bacteria that includes E. faecalis, E. faecium, E. durans, and E. avium. These bacteria are opportunistic pathogens that can cause urinary tract infections, wound infections, bacteremia, and endocarditis.

Another group of microorganisms that can be tested by the bile esculin test are non-spore-forming, hemolytic, Gram-positive rods that are catalase-positive and morphologically identified as presumptive Listeria. Listeria is a genus of bacteria that includes L. monocytogenes, L. ivanovii, L. innocua, and L. welshimeri. These bacteria can cause listeriosis, a serious infection that affects pregnant women, newborns, elderly people, and immunocompromised individuals.

The bile esculin test can also be used to rapidly identify enterococci and Listeria from positive blood cultures with Gram-positive cocci in chains or Gram-positive rods. The test can provide results within 4 hours of inoculation.

Finally, the bile esculin test can be modified by using esculin without bile for the identification of oxidase-positive aerobic Gram-negative rods, including Aeromonas and yellow-pigmented non-glucose-fermenting rods. These bacteria are waterborne pathogens that can cause gastroenteritis, wound infections, septicemia, and meningitis.

Media Used

Bile-esculin agar slants with iron(III) citrate. Agar plate media, such as Enterococcosel agar, have a similar formulation.

Bile-esculin-azide agar or broth with iron(III) citrate and azide. Azide will inhibit most Gram-negative bacteria.

Peptose-yeast-esculin broth (usually in the anaerobic atmosphere).

Esculin agar (0.1% esculin in heart infusion basal medium) without bile or azide but with iron(III) citrate.

The composition of Bile Esculin Agar is given below:

Reagents and Supplies Used

• Long-wave (360-nm) UV light
• 1% ferric ammonium citrate if iron(III) is not incorporated into the medium
• Sterile inoculating needle or loop
• Sterilized sticks, needles, or inoculating loops
• Pasteur pipettes
• Boiling heat block

The bile esculin test can be performed either by using a tube test or a disk test. The tube test is more commonly used and requires bile esculin agar slants with iron(III) citrate. The disk test is a rapid test that uses esculin disks moistened with water.

Tube test

1. Using a sterile loop, pick one or two colonies from an 18-24 hours culture of the organism to be tested.
2. Inoculate onto the surface of the slant of bile esculin medium with an S-shaped motion.
3. Incubate the inoculated tube at 35-37°C for 24 hours.
4. Observe the result by looking for the blackening of the medium.

Disk test

1. Moisten an esculin disk with a single drop of distilled or deionized water. Do not saturate the disk.
2. Using a sterile loop, pick two or three well-isolated colonies from an overnight (18- to 24-h) culture of the organism to be tested.
3. Inoculate the disk by rubbing the colonies on its surface.
4. Observe the result by looking for the development of a dark brown or black color after about 10 minutes at room temperature.

• The bile esculin test is not a definitive test for the identification of Enterococci and Group D Streptococci. It should be used in conjunction with other biochemical tests to confirm the identity of the isolated organism.
• Some viridans group streptococci (approximately 3%) may also hydrolyze esculin in the presence of bile. Therefore, the bile esculin test cannot be used to distinguish Streptococcus bovis from other viridans group streptococci without bile.
• Some other Gram-positive bacteria, such as Staphylococcus, Aerococcus, and Listeria monocytogenes, may grow in the presence of bile and hydrolyze esculin. Listeria monocytogenes will form minute black colonies on bile esculin agar.
• A heavy inoculum on bile esculin agar may cause a false-positive result by decreasing the ability of the bile to inhibit the growth of other Gram-positive organisms that may hydrolyze esculin.
• Some organisms may produce hydrogen sulfide during metabolism, which may react with iron and produce a black complex. This may interfere with the interpretation of the bile esculin test and give a false-positive result.
• Some bacteria, such as Escherichia coli, have β-glucosidase and will give a positive result in this test only after prolonged incubation. However, prolonged incubation should not be used if the test is being used to detect β-glucosidase in other organisms.
• As a result of nutritional requirements, some organisms may grow poorly or not at all on bile esculin agar.

Bile esculin test is a biochemical test that has several uses in microbiology, such as:

• It is used to presumptively identify Enterococci and Group D Streptococci on the basis of their ability to hydrolyze esculin in the presence of bile. These organisms are clinically important as they can cause infections such as endocarditis, bacteremia, urinary tract infections, and wound infections.
• It is used to differentiate these organisms from viridans Streptococci and other Gram-positive microorganisms that are either inhibited by bile or unable to hydrolyze esculin. Viridans Streptococci are a heterogeneous group of commensal bacteria that can also cause infections such as dental caries, subacute endocarditis, and abscesses.
• Bile Esculin Agar is a selective differential medium for the growth of organisms like Enterococcus, Listeria, and Yersinia enterocolitica. These organisms can grow in the presence of bile and produce a dark brown or black color on the agar due to esculin hydrolysis. Listeria and Yersinia are foodborne pathogens that can cause gastroenteritis, meningitis, septicemia, and abortion.
• Bile Esculin Test can also be used for the identification of oxidase-positive aerobic Gram-negative rods, such as Aeromonas and yellow-pigmented non-glucose-fermenting rods. These organisms can hydrolyze esculin without bile and produce a loss of fluorescence or a black color after adding ferric ammonium citrate. Aeromonas can cause diarrhea, wound infections, and septicemia in humans.

Bile Esculin Test is a simple, rapid, and inexpensive test that can provide useful information for the identification and differentiation of various microorganisms. However, it should be used in conjunction with other biochemical tests to confirm the identity of the isolated organism.

• The bile esculin test is not a definitive test for the identification of Enterococci and Group D Streptococci. It should be used in conjunction with other biochemical tests and morphological characteristics to confirm the identity of the isolates.
• Some viridans group streptococci (approximately 3%) may also hydrolyze esculin in the presence of bile. Therefore, the bile esculin test cannot be used to distinguish Streptococcus bovis from other viridans group streptococci without bile.
• Some other Gram-positive bacteria, such as Staphylococcus, Aerococcus, and Listeria monocytogenes, may grow in the presence of bile and hydrolyze esculin. Listeria monocytogenes will form minute black colonies on bile esculin agar.
• A heavy inoculum on bile esculin agar may cause the interpretation of the test to be difficult. Excess inoculum may decrease the ability of the bile to inhibit the growth of other Gram-positive bacteria that may hydrolyze esculin.
• Some bacteria may produce hydrogen sulfide during metabolism, which may react with iron and produce a black complex. This may interfere with the results of the bile esculin test and give a false-positive result.
• Some bacteria, such as Escherichia coli, have β-glucosidase and will give a positive result in this test only after prolonged incubation. However, prolonged incubation should not be used if the test is being used to detect β-glucosidase in other bacteria.
• As a result of nutritional requirements, some organisms may grow poorly or not at all on bile esculin agar.

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