What is the Bile Esculin Test?

In a biochemical analysis known as the Bile Esculin test, the ability to hydrolyze esculin permits Enterococci and group D Streptococci to be separated from non-group D viridans group Streptococci.

• Although several organisms are capable of hydrolyzing esculin,only a few number of them can do it when bile is present (4 percent bile salts or 40 percent bile.). Consequently, this trait is utilized to categorise species into several groupings.
• On bile esculin agar, a selective differential agar containing both bile and esculin, the bile-esculin experiment is performed.
• Different bile salts found in the agar prevent the development of additional Gram-positive organisms as well asenable the selective separation of enterococci and group D streptococci.
• Esculin is a luminous glycosidic coumarin derivative (6-beta-glucoside-7-hydroxy-coumarin), and the decrease of the fluorescence indicates the presence of hydrolysis.
• The bile-esculin examination has been refined and accelerated throughout time. Using bile-esculin discs, it is now able to swiftly differentiate among Group D Streptococci as well as non-Group D Streptococci.

Objectives of Bile Esculin Test

• Differentiate between Enterococci and Group D Streptococcus based on their ability to hydrolyze esculin in the existence of bile.
• to distinguish Group D Streptococci and Enterococci from other viridans or other types of Streptococci.

Principle of Bile Esculin Test

• Esculin is hydrolyzed in the existence of bile salt by the enzyme esculinase, which forms the foundation of the esculin test.
• Esculin is a glucoside made of hydroxycoumarin and glucose that is joined by an ester bond with oxygen.
• The bile esculin analysis selects organisms depending on their ability to thrive in a medium with 4% bile salts, and afterwards selects organisms having the ability to hydrolyze esculin.
• Esculin undergoes hydrolysis to generate glucose and esculetin.
• After esculin breakdown, the esculetin produced by the hydrolysis of esculin mixes with iron ions (from ferric citrate) as in medium to form a phenolic iron complex, which imparts a dark brown or black colour to the substance.
• In contrast, esculin is a fluorescent chemical. and a reduction of fluorescence after hydrolysis may be seen.
• The bacteria must be capable of thriving in the influence of bile,if it is given to the medium in order for it to hydrolyze esculin. The bile prevents the development of other
• Gram-positive bacteria, which increases the medium’s level of selection.
• The majority of Streptococci, except Streptococcus bovis, are inhibited by the bile esculin medium’s 40% bile content (equal to 4% oxgall), however Enterococci and Listeria are not.

Microorganism tested

• Gram-positive chains of cocci that lack the catalase enzyme and are visually recognised as supposing to be S. bovis.
• Being part of the procedure for separating enterococci from other pyrrolidonyl-naphthylamide (PYR)-positive organisms, pyrrolidonyl-naphthylamide (PYR) levels are measured. isolates of alpha- or gamma-hemolytic, Gram-positive cocci are made.
• Gram-positive, hemolytic, non-spore-forming rods that have been morphologically characterised as probable Listeria and are also catalase-positive
• Gram-positive cocci in chains or Gram-positive rods in positive blood cultures can quickly (within 4 hours) detect enterococci and Listeria
• The use of esculin without bile to identify oxidase-positive aerobic
• Aeromonas and yellow-pigmented non-glucose-fermenting rods, among other gram-negative rods

Media, Reagents, and Supplies Used

Media Used

• Iron(III) citrate slants in bile-esculin agar. Agar plate media have a similar composition as Enterococcisel agar.
• Agar or broth with iron(III) citrate and azide, together with bile-esculin. Most Gram-negative bacteria will be inhibited by azide.
• Esculin-yeast-peptose broth (usually in the anaerobic atmosphere).
• Esculin agar contains iron(III) citrate but no bile or azide (0.1 percent esculin in cardiac infusion basal medium).

Below is a list of the ingredients in bile esculin agar:

Reagents and Supplies Used

• 360-nm long-wave UV light
• iron(III) with a 1% ferric content. If iron(III) is not integrated into the medium, use ammonium citrate.

The procedure of Bile Esculin Test

Preparation of media

• 5 grammes of dehydrated powder or laboratory-prepared media are added to a beaker holding 1000 millilitres of deionized or distilled water.
• The liquid is then brought to a boil in order to completely dissolve the powder.
• The dissolved medium is subsequently split among tubes and sterilised in an autoclave for 15 minutes at 121°C and 15 pounds of pressure.
• Following the autoclaving process is complete, the tubes are withdrawn and cooled to about 40–45°C while kept in an inclined position. Maintaining the posture is necessary to get butts that are 1.5 to 2.0 cm deep.

Esculin Hydrolysis

• You can detect esculin hydrolysis using a disc test or a tube test. An expedient test is a disc test.

Tube test

• Using a sterile inoculating needle, a well-isolated colony is extracted from an 18–24 hour culture.
• The slant is infected by stippling the surface either with light inoculum picked from the culture dish or the bile esculin agar tubes.
• For the detection of enterococcus and S. bovis, the tubes are infected with a 10-l calibrated loop of a 0.5 McFarland standard solution produced in sterile water.
• To ensure adequate aeration, the test tube tops should be removed.
• The colour shift is then seen after the tubes have been incubated aerobically at 35–37°C for 24 hours (or up to 7 days for slow-growing Gram-negative rods and anaerobes).
• The tubes for esculin broth without iron (III) citrate are checked for loss of fluorescence every day.
• In the exclusion of fluorescence, two or three drops of ferric ammonium citrate at a concentration of 1 percent are added to the esculin tube, and the colour change is seen.

Disk test

• The esculin disc is wetted with one drop of deionized or distillated water. However, the disc must not become saturated.
• Using a sterile loop, two to three well-isolated colonies are picked from an overnight (18 to 24-hour) culture.
• Following around 10 minutes at ambient temperature, the disc is checked to see whether a dark brown or black hue has developed.

Result Interpretation of Bile Esculin Test

• A symptom is that the medium becomes black after a positive tube test in medium having ferric ammonium citrate.
• The absence of colour change signifies a negative tube test. The substance will glow under UV light (366 nm).
• A positive test for esculin broth absent iron (III) citrate is indicated by the blackening of the broth upon addition of the ferric [iron(III)] reagent or by the loss of fluorescence of the medium.
• Whether or not the organism can hydrolyze esculin, a negative test result also happens in the bile-esculin medium if it is unable to develop when bile is present.
• A positive disc test is indicated by the emergence of a dark brown or black colour.
• The test result for the colourless disc is negative.

The following table depicts the growth of several bacteria as well as their bile esculin hydrolysis test.

Uses of Bile Esculin Test

• The bile esculin test is a biochemical procedure utilized to extract enterococci as well as group D streptococci.
• These organisms may be distinguished from viridans Streptococci and other Gram-positive germs using this method as well.
• Bile esculin agar is a selective differential medium for the development of microorganisms such as Enterococcia, Listeria, and Yersinia enterocolitica.

Limitations of Bile Esculin Test

• Streptococci other than S. aureus are included in the viridans group if a substantial inoculum is utilised or if the content of bile is less than 40%. Bovis could respond well to bile-esculin agar.
• Esculin tests done without bile are unable to differentiate S. bovis from other viridans group streptococci. Bovis was formerly known as group D streptococci.
• The esculin hydrolysis test may generate false-positive findings because during metabolism, certain organisms may emit H2S, which may mix with iron to create a black complex.\
• Some microbes, including E. Only after a lengthy incubation period can E. coli that produce -glucosidase provide a positive result in this test. However, if the test is being used to find -glucosidase in other species, lengthy incubation shouldn’t be performed.

References and Sources

• Bile esculin agar. M1225. HiMedia Laboratories.
• Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook, 3.17.1.1–3.17.48.3.DOI:10.1128/9781555818814.ch3.17.1
• Chuard, L. B. Reller. Bile-Esculin Test for Presumptive Identification of Enterococci and Streptococci: Effects of Bile Concentration, Inoculation Technique, and Incubation Time. Journal of Clinical Microbiology Apr 1998, 36 (4) 1135-1136; DOI: 10.1128/JCM.36.4.1135-1136.1998.
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