Bile Esculin Test: Objective, Principle, Procedure, Result, Uses, Limitations

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What is Bile Esculin Test ?

The bile esculin test is a biochemical test that uses the capacity to hydrolyzeesculin to distinguish Enterococci and group D Streptococci from non-group D viridans group Streptococci.

Although many species can hydrolyzeesculin, only a handful can do it in the presence of bile (4 percent bile salts or 40 percent bile.). As a result, this feature is used to identify organisms belonging to a specific group.
The Bile-esculin test is performed on bile esculin agar, a selective differential agar that contains both bile and esculin.
The agar contains several bile salts that prevent the growth of other Gram-positive organisms, allowing Enterococci and Group D Streptococci to be isolated selectively.
Esculin is a luminous glycosidiccoumarin derivative (6-beta-glucoside-7-hydroxy-coumarin), and its hydrolysis can also be seen by the loss of fluorescence.
Through the years, the bile-esculin test has been improved and made more quick. Bile-esculin discs are now widely utilised to distinguish between Group D Streptococci and non-Group D Streptococci.

Objectives of Bile Esculin Test

To find the ability of Enterococci and Group D Streptococci to hydrolyzeesculin in the presence of bile.
To distinguish Enterococci and Group D Streptococci members from other viridans and non-Group D Streptococci.

Bile Esculin Test Principle

The esculin test is based on the hydrolysis of esculin in the presence of bile salt due to esculinase’s enzymatic action.
Esculin is a glucoside made up of glucose and hydroxycoumarin connected by an ester bond in the presence of oxygen.
The bile esculin test first selects organisms based on their capacity to grow in a medium containing 4% bile salts, then selects organisms based on their ability to hydrolyzeesculin.
When esculin is hydrolyzed, glucose and a molecule called esculetin are produced.
Following esculin degradation, the esculetin formed by esculin hydrolysis combines with iron ions (from ferric citrate) in the medium to form a phenolic iron complex, giving the product a dark brown or black hue.
Esculin, on the other hand, is a fluorescent chemical, and its hydrolysis is visible as a loss of fluorescence.
In order to hydrolyzeesculin, the microbe must be able to grow in the presence of bile in the medium. Other Gram-positive organisms are inhibited by the bile, making the medium more selective.
The bile esculin medium contains 40% bile (equal to 4% oxgall), which inhibits most Streptococci strains except Streptococcus bovis, but not Enterococci or Listeria.

Microorganism Tested

Gram-positive cocci that are catalase-negative and morphologically identified as presumptive S. bovis in chains.
As part of the distinction of enterococci from other pyrrolidonyl—naphthylamide (PYR)-positive organisms, isolates of alpha- or gamma-hemolytic, Gram-positive cocci were obtained.
Presumptive Listeria are nonspore-forming, hemolytic, Gram-positive rods that are catalase-positive and morphologically identifiable.
Positive blood cultures containing Gram-positive cocci in chains or Gram-positive rods to diagnose enterococci and Listeria quickly (within 4 hours).
For the identification of oxidase-positive aerobic Gramnegative rods, such as Aeromonas and yellow-pigmented non-glucose-fermenting rods, Esculin without bile is used.

Media, Reagents, and Supplies Used

Media Used

Iron(III) citrate slants bile-esculin agar. Agar plate media with a similar formulation, such as Enterococcosel agar, are available.
Agar containing bile, esculin, and azide, or broth containing iron(III) citrate and azide. Most Gram-negative bacteria are inhibited by azoide.
Broth with peptose, yeast, and esculin (usually in the anaerobic atmosphere).
Esculin agar (0.1 percent esculin in cardiac infusion basal medium) containing iron(III) citrate but no bile or azide.

The following is a list of the ingredients in Bile Esculin Agar:

[ninja_tables id=”3904″]

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Reagents and Supplies Used

UV radiation with a wavelength of 360 nanometers.
1 % ferric [ferrous(III)] if the medium does not contain iron(III), ammonium citrate.

Procedure of Bile Esculin Test

Preparation of Media

64.5 grammes of dehydrated powder or lab-prepared media are mixed with 1000 millilitres deionized or distilled water in a beaker.
The medium is then brought to a boil to completely dissolve the powder.
The dissolved medium is then poured into tubes and sterilised in an autoclave for 15 minutes at 15 lbs pressure (121°C).
After the autoclaving process is completed, the tubes are removed and cooled to a temperature of around 40-45°C in a tilted posture. To obtain butts of 1.5–2.0 cm depth, the posture should be maintained.

Esculin Hydrolysis

A tube test or a disc test can be used to detect esculin hydrolysis. A disc test is a quick examination.

Tube test

With the use of a sterile inoculating needle, a well-isolated colony is extracted from an 18-24 hour culture.
The light inoculum from the culture plate is streaked across the surface of the bile esculin agar tubes.
For enterococcus and S. bovis identification, a 10-l calibrated loopful of a 0.5 McFarland standard suspension prepared in sterile water is used, and the tubes are inoculated with a 10-l calibrated loopful of a 0.5 McFarland standard suspension prepared in sterile water.
To maintain appropriate aeration, the test tube caps should be left unfastened.
After that, the tubes are incubated aerobically at 35-37°C for 24 hours (or up to 7 days for Gram-negative rods and anaerobes that develop slowly), and the colour change is noticed.
The tubes in the esculin broth without iron (III) citrate are checked daily for decrease of fluorescence.
2 or 3 drops of 1.0 percent ferric ammonium citrate are added to the esculin tube if there is no fluorescence.

Disk test

A single drop of distilled or deionized water is used to moisten the esculin disc. However, the disc should not be saturated.
Two to three well-isolated colonies are selected from an overnight (18 to 24-hour) culture using a sterile loop.
After about 10 minutes at room temperature, the disc is examined for the formation of a dark brown or black tint.

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Result Interpretation of Bile Esculin Test

The blackening of the medium indicates a positive tube test in medium containing ferric ammonium citrate.
A lack of colour change indicates a negative tube test. Under UV light, the medium will glow (366 nm).
A positive test for esculin broth without iron (III) citrate is shown by the broth turning black after adding the ferric [iron(III)] reagent or the medium losing its fluorescence.
In the bile-esculin medium, a negative test result can also occur if the organism cannot grow in the presence of bile, independent of its capacity to hydrolyzeesculin.
The development of a dark brown or black colour on the disc indicates a positive disc test.
A colourless disc test results in a negative result.

The development of various bacteria and their bile esculin hydrolysis test are shown in the table below:

[ninja_tables id=”3906″]

Uses of Bile Esculin Test

The bile esculin test is used to isolate Enterococci and Group D Streptococci as a biochemical test.
It can also be utilised to distinguish these organisms from Streptococci viridans and other Gram-positive bacteria.
Enterococcia, Listeria, and Yersinia enterocolitica can all grow on bile esculin agar, which is a selective differential media for their growth.

Limitations of Bile Esculin Test

Other viridans group streptococci, such as S. bovis, may produce a positive reaction on bile-esculin agar if a large inoculum is used or if the bile concentration is less than 40%.
S. bovis (previously known as group D streptococci) and other viridans group streptococci cannot be distinguished using esculin tests without bile.
Several species create H2S during metabolism, which reacts with iron to form a black complex, interfering with the results of the esculin hydrolysis test and potentially leading to a false-positive result.
Some microorganisms with -glucosidase, such as E. coli, will only give a positive result in this test after a long period of incubation.

Bile Esculin Test Citations

https://jcm.asm.org/content/36/4/1135
https://clinicalgate.com/gram-positive-cocci/
http://www.austincc.edu/microbugz/bile_esculin_test.php
http://microsc.net/materials/4net/laboratory_syllabus/use_of_selective_and_differential_media.pdf
https://vlab.amrita.edu/?sub=3&brch=73&sim=703&cnt=2
https://pubchem.ncbi.nlm.nih.gov/compound/fluorescein
https://persianlab.com/strepiococcus-enterococcus-and-other-catalase-negative-gram-positive-cocci/
https://en.wikipedia.org/wiki/Bile_esculin_agar
https://www.sciencedirect.com/topics/medicine-and-dentistry/streptococcus-group-d
https://www.coursehero.com/file/p610isqs/Alternatively-esculin-is-a-fluo-rescent-compound-and-its-hydrolysis-can-be/
https://www.bd.com/resource.aspx?IDX=8992
https://en.wikipedia.org/wiki/Agar_plate
https://biologicalindicators.mesalabs.com/wp-content/uploads/sites/31/2014/02/Unique-Cycles-Sterilizing-Liquid-Loads.pdf
https://asm.org/Articles/2020/January/Microbiology-Laboratory-Tips-and-Tricks-An-Organis

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