The bile esculin agar It is a selective and differential solid culture medium. It is used as a diagnostic test to determine the ability of a certain microorganism to grow in a medium containing bile and also break down the glucoside esculin into esculetin and glucose..
This diagnostic test is used to differentiate species of the genus Streptococcus belonging to group D (bile esculin positive), from other groups of Streptococcus that react negatively to this test..
It should be noted that some Streptococcus of the viridans group can hydrolyze esculin, but they are not capable of growing in the presence of bile at a concentration of 40%, therefore, in this medium the reaction for this group is negative.
On the other hand, the bile esculin medium is also useful for the diagnosis of Listeria monocytogenes or species of Aerococcus sp, since these microorganisms are bile esculin positive.
Esculin bile agar is composed of peptone, meat extract, ox bile, esculin, iron citrate, agar, and distilled water. Some commercial houses include sodium azide within the composition of the medium.
The medium can be prepared in the laboratory if you have all the compounds separately or it can be prepared from the commercial dehydrated medium..
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The bile esculin medium contains peptones and meat extract, both compounds provide the necessary nutrients for the growth of microorganisms..
It also contains esculin; This compound is a glycoside formed by the union of a simple monosaccharide (glucose) with a compound called 6,7-dihydroxycoumarin or esculetin (aglucone), linked by an acetal or glucosidic bond.
The test is based on showing whether the bacteria is capable of hydrolyzing esculin. If this happens, the esculin breaks down into esculetin and glucose. Esculetina reacts with the iron present in the medium, forming a dark brown, almost black compound.
This means that ferric citrate acts as a reaction developer. This characteristic makes bile esculin agar a differential medium..
For its part, bile is an inhibitor that prevents the growth of some microorganisms, therefore, the bacteria before splitting the esculin must be able to grow in the presence of bile. Therefore, this medium is considered selective.
The bacteria that can develop in this environment are mainly those that live in the intestinal environment.
In this sense, some commercial companies add sodium azide to the medium to further inhibit the growth of enteric Gram negative bacilli, increasing the selectivity of the medium for the growth of Streptococcus..
Finally, the agar gives the solid consistency to the medium and water is the solvent of the compounds..
Weigh:
5 g peptones
3 g of meat extract
40 g beef bile
1 g of esculin
0.5 g of iron citrate
15 g agar
1000 ml of distilled water
In case of adding sodium azide, weigh 0.25 g / liter and add to the mixture.
Dissolve the components in the liter of distilled water, heat until the compounds are completely dissolved. Dispense 5 ml into 16 x 125 mm screw cap test tubes. Autoclave at 121 ° C, 15 pounds pressure for 15 minutes.
Remove from the autoclave and tilt the tubes on a support, so that the agar solidifies in a wide flute beak.
Store in a refrigerator until use. Bring to room temperature before sowing.
Bile esculin agar plates can also be prepared; in this case, the entire mixture is autoclaved in a flask and subsequently distributed in sterile Petri dishes. They are left to solidify and stored in the fridge..
The pH of the medium must be 6.6 ± 0.2.
Weigh the amount specified by the insert. This can vary from one business house to another. Subsequently, proceed the same as the procedure explained above.
The pH of the medium should be 6.6 ± 0.2. The color of the dehydrated medium is light beige and the prepared medium is dark amber.
The bile esculin medium is mainly used to differentiate Group D Streptococcus (bile esculin positive), from the rest of the Streptococcus groups (bile esculin negative).
Combining the hypersalted broth growth test with the bile esculin test can identify a special group of group D Streptococcus called Enterococcus.
This special group of Streptococcus belongs to group D of the mentioned genus and they are capable of hydrolyzing esculin in the presence of bile as do the rest of the members of group D, but they are also capable of developing in a hypersalted medium (BHI with chloride of 6.5% sodium), a property that makes the difference.
Therefore, Streptococci that hydrolyze esculin bile but do not grow in hypersalted broth are called non-enterococci Group D Streptococci..
Inoculate the medium preferably from a pure Todd-Hewitt 24-hour broth.
Add 2 drops to the surface of the medium with a Pasteur pipette and spread in the medium with a platinum loop..
Incubate at 35 ° C for 48 hours, while the incubation time is met, it can be monitored to see if there is a positive reaction. If at the end of the time the reaction is still negative, it can be incubated for up to 72 hours.
Positive reaction: Appearance of a dark brown, almost black color in the flute beak (in the case of the tube test) or blackening of the agar around the colonies (in the case of the plate test).
Negative reaction: no blackening of the medium or black appears in less than half of the tube after 72 hours of incubation. On the other hand, bacterial growth in the medium without the appearance of the black color should be considered a negative test..
To evaluate the quality of the medium, a strain of Enterococcus faecalis ATCC 29212 as a positive control and a non-group D Streptocococcus strain as a negative control.
-Media that does not contain sodium azide allow the growth of enteric Gram negative rods. Some of them can blacken the middle.
- Some commercial houses add low concentration of bile (10%) and for this reason some Streptococcus that do not belong to group D can develop in the medium and hydrolyze esculin, which can generate errors in interpretation..
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