Bacteria that are bile-esculin positive are able to grow in the presence of bile salts and the hydrolysis of the esculin in the medium results in the formation of glucose and a compound called esculetin. Esculetin, in turn, reacts with ferric ions (supplied by the inorganic medium component ferric citrate
Citric acid
Citric acid is a weak organic acid that has the chemical formula C₆H₈O₇. It occurs naturally in citrus fruits. In biochemistry, it is an intermediate in the citric acid cycle, which occurs in the metabolism of all aerobic organisms.
What is bile esculin agar used for?
Uses of Bile Esculin Agar It is recommended for use as a differential medium in the isolation and presumptive identification of enterococci/group D streptococci. It is used in conjunction with other biochemical tests to identify cultures of isolated organism.
What is the role of oxgall in BEA and how it works?
, what is the role of oxgall in BEA and how does it work? The medium contains bile that inhibit the growth of gram positive bacteria an the sodium azide inhibits the growth of gram negative bacteria. Esculin is a carbohydrate linked to alcohol, several organisms can hydrolyze esculin, but only selective organisms do this in the presence of bile
What is the effect of inoculum on the BEA test?
A heavy inoculum on BEA may cause interpretation of the bile esculin test difficult to read. Excess inoculum decreases the ability of the bile to inhibit the growth of other gram-positive organisms that may hydrolyze esculin. There are a few streptococci that do not hydrolyze esculin but will grow in the presence of bile.
What is the history of esculin hydrolysis?
Esculin hydrolysis was first described by Rochaix in 1924. Swan first introduced the use of Bile Esculin Agar in 1954. Bile Esculin Agar (BEA) is a selective differential agar used to isolate and identify members of the genus Enterococcus, formerly part of the “group D streptococci”.
What is esculin test used for?
This test is used for the presumptive identification of enterococci and organisms in the Streptococcus bovis group. The test differentiates enterococci and group D streptococci from non–group D viridans streptococci.
How does the bile-esculin test work?
The bile esculin test selects organisms first on the basis of their ability to grow in a medium with 4% bile salts followed by the selection based on their ability to hydrolyze esculin. The hydrolysis of esculin results in glucose and a compound called esculetin.
What is BEA agar selective for?
Bile Esculin Agar (or Enterococcal Agar or BEA) is prepared according to the formula specified by Swan, and subsequently evaluated by Facklam and Moody. It is a selective medium for the isolation of Enterococci and Group D Streptococci.
What Streptococcus are bile-esculin positive?
All strains of S. bovis and approximately 10% of viridans streptococci are bile-esculin positive.
What enzyme does bile esculin agar test for?
Bile esculin test is based on the hydrolysis of esculin into glucose and esculetin (6, 7-dihydroxy-coumarin) by a micro-organism that produce an enzyme esculinase.
What is the substrate in bile esculin agar?
p-Nitrophenyl-beta-D-glucopyranoside was the esculinase substrate, and sodium desoxycholate was substituted for bile salts. After inoculation, a yellow color was equivalent to the brown-black seen in the 40% bile-esculin reaction.
What is BBE agar?
Bacteroides Bile Esculin (BBE) agar is an enriched, selective, and differential medium used for the isolation and presumptive identification of obligately anaerobic gram-negative bacilli of the Bacteroides fragilis group and Bilophila wadsworthia. BBE Agar Plate.
What ingredients make BEA a selective medium?
Bile Esculin Agar (BEA) is a selective differential agar used to isolate and identify members of the genus Enterococcus, formerly part of the “group D streptococci”....Composition of Bile Esculin Agar.IngredientsGms/literBeef extract3.00Esculin1.00Bile salts40.0Ferric citrate0.502 more rows•Jan 3, 2022
What are the nutrients in bile esculin agar?
Bile esculin agar contains oxgall (bile salts) to inhibit the growth of gram positive organisms other than enterococci and group D streptococci. It also contains nutrients, esculin, and ferric citrate.
Why is bile-esculin used?
The bile-esculin test is used to differentiate enterococci and group D streptococci from non-group D viridans group streptococci.
Why is BEA used when a streptococcal infection is suspected?
It is commonly used to identify members of the genus group D streptococci and enterococci. BEA medium contains bile to inhibit the growth of gram-positive bacteria as well as many gram-negative bacteria, and esculin which is the differential component.
What color does the membrane turn if a bacteria is positive for esculin hydrolysis?
In slants, positive result is when more than half the medium is blackened. Spot inoculation. If an organism can hydrolyze esculin, then the media will turn dark brown or black. More than half the medium will be dark brown or black after incubation (in slants).
Is bile Esculin selective or differential?
This is a medium that is both selective and differential. It tests the ability of organisms to hydrolyze esculin in the presence of bile.
How is Phenylethanol agar pea considered selective?
Phenylethyl alcohol agar (PEA) is a selective medium used to cultivate Gram positive organisms. The active ingredient, phenylethyl alcohol, inhibits or markedly reduces growth of Gram negative organisms by interfering with DNA synthesis. PEA also prevents Proteus species from swarming across the surface of the agar.
What is the selective agent found in MSA?
Mannitol Salt Agar (MSA) This is a medium that is both selective and differential. The high salt concentration (7.5%) is the selective ingredient. Staphylococcus species, which commonly inhabit human skin, can grow on this high salt concentration (left plate in picture below).
Is TSA selective or differential?
Tryptic Soy Agar (TSA): a general purpose, non-selective, non-differential, supportive medium that supports growth of all microorganisms that do not require special nutrients.
What is bile esculin agar used for?
Bile Esculin agar (BEA) is used for this test. BEA is a selective and differential medium which is presumptively used to identify Enterococci and group D Streptococci based on the ability of an organism to hydrolyze esculin. Bile esculin agar contains oxygall (bile salts, first selective ingredients) to inhibit the growth of other gram positive organisms other than Enterococci and group D Streptococci. It contains sodium azide (second selective ingredients) to inhibit the growth of gram negative organisms. It also contains nutrients esculin and ferric citrate. Esculin (differential ingredient) is a fluorescent compound and its hydrolysis can be observed by a loss of fluorescence.#N#Bile esculin disk is used for the rapid detection of esculin hydrolysis in presence of bile for differentiating group D Streptococci from non-group D Streptococci.
What is the reaction of esculin and iron salt?
Esculetin reacts with an iron salt (ferric citrate) in the medium to form a phenolic iron complex which produces a dark brown or black color. Esculin + Acid → β-D-glucose + Esculetin. Esculetin + Fe3+ → Dark Brown Color.
How long to incubate bile esculin?
Inoculate onto the surface of slant of bile esculin medium with an S-shaped motion. Incubate the inoculated tube at 35-37°C for 24 hours. Observe the result.
Can Escherichia coli react with a glucosidase test?
Limitation. Some micro-organisms such as Escherichia coli have a β-glucosidase and will react in this test after prolonged incubation. Prolonged incubation should not be used if the test is being used to detect only constitutive β-glucosidase.
Can a Viridans group streptococci give a positive reaction on bile escul?
If the inoculum is too great or the concentration of bile is less than 40%, viridans group streptococci other than S.bovis can give a positive reaction on bile-esculin agar. A heavy inoculum on BEA may cause interpretation of the bile esculin test difficult to read.
Principle
Gram-positive bacteria other than some streptococci and enterococci are inhibited by the bile salts in this medium. Organisms capable of growth in the presence of 4% bile and able to hydrolyze esculin to esculetin. Esculetin reacts with Fe 3+ and forms a dark brown to black precipitate.
Method
Inoculate one to two colonies from an 18- to 24-hour culture onto the surface of the slant.
Uses
This test is used for the presumptive identification of enterococci and organisms in the Streptococcus bovis group.
Limitations
It should be used in conjunction with other biochemical tests to identify cultures of isolated organism.
How does colistin work?
These cationic regions in colistin work by interaction with bacterial outer membrane, and function by displacing calcium and magnesium bacterial counter ions in the lipopolysaccharide. Hydrophobic/hydrophilic regions hence it interacts with the membrane like a detergent does , it thus solubilizes the cytoplasmic membrane.
What is Ox Gall?
OX gall is dehydrated bile sed in preparing microbiological culture media. The major composition of ox gall is taurocholic and glycocholic acids and it provides bile salts and only few bacterial species like group D streptococci and Enterococcus tolerate bile salts and can hydrolyze esculin to esculetin.
What is a substrate?
1. Substrate (s) such as a carbohydrate that only certain bacterial species can utilize in a specific chemical reaction or a set of chemical reactions
Does solistin work on Gram negative strains?
Since most gram positive bacteria dont have outer membrane hence solistin has nothing to interact much hence it works mostly for gram negative strains than gram positive strains.
Is Ox Gall a selective agent?
Ox gall is used as a selective agent for the isolation of gram-negative micro-organims as they can only tolerate bile salts, inhibiting the growth of gram-positive micro-organisms as they cannot tolerate it and the cells are destroyed in its presence .
Principle
Procedure
- A. Tube Test
1. Using sterile loop, pick one or two colonies from an 18-24 hours culture. 2. Inoculate onto the surface of 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. - B. Disk Test
1. Moisten disk with a single drop of distilled water. 2. Using a sterile loop, pick two or three well-isolated colonies from an overnight culture. 3. Inoculate on the disk. 4. Allow to react at room temperature for 10min. 5. Observe the disk for the development of a dark brown or black color.
Result Interpretation
- Positive: Blackening of more than half of the agar slant, if the medium containing ferric ammonium citrate is used. NOTE: In case of esculin broth without iron citrate, positive test means blackening of the broth after addition of the ferric reagent or by the loss of fluorescence of the medium. Negative:No blackening of medium. OR If the agar slant is less than half darkened or m…
Quality Control
- Bile esculin positive:Enterococcus faecalis ATCC 29212 Bile esculin Negative:Streptococcus pyogenes ATCC 19615
Limitation
- Some micro-organisms such as Escherichia colihave a β-glucosidase and will react in this test after prolonged incubation. Prolonged incubation should not be used if the test is being used to detect...
- H2S which is produced by several organisms during metabolism, also reacts with iron to produce black complex which interferes with the interpretation of the esculin hydrolysis test.
- Some micro-organisms such as Escherichia colihave a β-glucosidase and will react in this test after prolonged incubation. Prolonged incubation should not be used if the test is being used to detect...
- H2S which is produced by several organisms during metabolism, also reacts with iron to produce black complex which interferes with the interpretation of the esculin hydrolysis test.
- If the inoculum is too great or the concentration of bile is less than 40%, viridans group streptococci other than S.bovis can give a positive reaction on bile-esculin agar.
- A heavy inoculum on BEA may cause interpretation of the bile esculin test difficult to read. Excess inoculum decreases the ability of the bile to inhibit growth of other gra...
Principle
Method
- Inoculate one to two colonies from an 18- to 24-hour culture onto the surface of the slant.
- Incubate at 35°-37°C in ambient air for 48 hours.
- Observe for growth and blackening of the medium.
Expected Results
- Positive:Growth and blackening of the agar slant
- Negative: Growth and no blackening of medium ; No growth
Uses
- This test is used for the presumptive identification of enterococci and organisms in the Streptococcus bovis group.
- The test differentiates enterococci and group D streptococci from non–group D viridans streptococci.
Limitations
- It should be used in conjunction with other biochemical tests to identify cultures of isolated organism.
- As a result of nutritional requirements, some organisms may grow poorly or not at all on this medium.
- Some strains of Staphylococcus, Aerococcus, and Listeria monocytogenes may grow in the …
- It should be used in conjunction with other biochemical tests to identify cultures of isolated organism.
- As a result of nutritional requirements, some organisms may grow poorly or not at all on this medium.
- Some strains of Staphylococcus, Aerococcus, and Listeria monocytogenes may grow in the presence of bile and hydrolyze esculin. monocytogenes will form minute black colonies.
- A heavy inoculum on BEA may cause interpretation of the bile esculin test difficult to read. Excess inoculum decreases the ability of the bile to inhibit growth of other gram-positive organisms tha...
References
- Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
- https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/BileEscAgar.htm
- https://www.austincc.edu/microbugz/bile_esculin_test.php
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