Bile Esculin Agar: Recommended Procedure

BILE ESCULIN AGAR
- For in vitro use only - Catalogue No. PB65 & TB65
Our Bile Esculin Agar (BEA) is used as a Recommended Procedure

differential medium for the isolation and
presumptive identification of group D streptococci Enterococci & Streptococci (Group D)
and enterococci from clinical specimens and food. 1. Allow medium to reach room temperature.
This medium is also useful for differentiating 2. From a primary plate, pick suspect isolates of
Klebsiella, Enterobacter, and Serratia species from enterococci and perform a four-quadrant streak
other Enterobacteriaceae. onto a Bile Esculin Agar plate. Alternatively,
Swan was the first to describe the formulation from a pure, overnight culture of streptococci
and use of a bile esculin medium, although or enterococci grown in Todd-Hewitt broth,
Rochaix (1924) was the first to note the value of add 2 drops onto the agar surface and perform
using esculin hydrolysis to identify enterococci. a four-quadrant streak for maximum isolation.
During their comparative studies, Facklam and If inoculating a Bile Esculin Agar Slant follow
Moody demonstrated that Bile Esculin Agar was a the same procedure but streak the surface of
reliable method of presumptively identifying group the slant in a fish-tail motion.
D streptococci and differentiating them from other 3. Incubate aerobically at 35°C.
streptococci. Lindell and Quinn as well as 4. Examine after 18-24 hours for esculinase-
Edberg’s group showed that Bile Esculin Agar positive colonies.
could also be used for the rapid differentiation of 5. Hold up to 72 hours before reporting as
Enterobacteriaceae based on esculin hydrolysis negative.
later showed it.
Gelatin peptone and beef extract provide the Enterobacteriaceae
essential elements needed for growth. The 1. Allow medium to adjust to room temperature
inclusion of esculin allows for detection of esculin- 2. Take a heavy inoculum from a confirmed
hydrolysis by the bacterial enzyme, esculinase. gram-negative pure culture grown on heart
Esculin hydrolysis liberates esculetin, which in infusion agar, Lysine Iron Agar, or Triple
turn reacts with ferric ions (ferric citrate) in the Sugar Iron Agar and perform a four-quadrant
medium to produce a black iron-complex giving streak to maximize recovery of isolated
esculinase-positive colonies a brown-black halo. colonies.
Selectivity is accomplished by the addition of bile 3. Incubate aerobically at 35°C.
(oxgall), which inhibits the growth of most gram- 4. Examine after 18-24 hours.
positive cocci other than enterococci and group D
streptococci.
Interpretation of Results
Formula per Litre of Medium
Characteristically, group D streptococci and
Gelatin Peptone ................................................ 5.0 g enterococci grow in the presence of bile and
Beef Extract ...................................................... 3.0 g hydrolyze esculin. On Bile Esculin Agar, typical
Bile (Oxgall)................................................... 40.0 g group D streptococci and enterococci colonies
Esculin .............................................................. 1.0 g appear as small transparent colonies with brown-
Ferric Citrate .................................................... 0.5 g black halos. If these colonies are observed then
Agar ................................................................ 15.0 g this is a presumptive positive for enterococci.
pH 6.6 ± 0.2
To differentiate between enterococci and Organism Expected Result
group D streptococci a PYR disk test (Dalynn Enterococcus faecalis Growth with
DP95) and salt tolerance test (Dalynn TS27) can be ATCC 29212 blackening of medium
performed. All enterococci are PYR positive and
can grow in 6.5% NaCl (salt tolerance test) while Streptococcus pyogenes Inhibition
group D streptococci are negative for both tests. ATCC 19615
As mentioned, Bile Esculin Agar can be used
as a rapid test for differentiating
Enterobacteriaceae. Among the family, Klebsiella, Storage and Shelf Life
Enterobacter, and Serratia species are typically
esculinase-positive and hydrolyze esculin, while Our Bile Esculin Agar should be stored at 4°C
most other Enterobacteriaceae are esculinase- to 8°C and protected from light. The medium side
negative. should be uppermost to prevent excessive
Further biochemical and/or serological tests accumulation of moisture on the agar surface.
should be performed on isolated colonies from Under these conditions this medium has a shelf life
pure culture to complete identification. of 11 weeks from the date of manufacture.
• This is only a presumptive test for enterococci

and some strains of Lactococcus, Leuconostoc, References
Pediococcus, and Vagococcus, which have
been isolated from human infections, may 1. Rochaix A. Milieux a leculine pour le
produce similar results on Bile Esculin Agar diagnostid differentieldes bacteries du groups
strepto-entero-pneumocoque. Comt Rend Soc
• Bile Esculin Agar is a differential medium and Biol 1924; 90:771-2.
not a primary plating medium; use Bile 2. Swan A. The use of bile-esculin medium and
Esculin Azide Agar (Enterococcosel Agar) if a if Maxted’s technique of Lancefield grouping
primary plating medium is desired in the identification of enterococci (group D
streptococci). J Clin Path 1954; 7:160.
• Approximately 3 % of viridans streptococci 3. Facklam RR, Moddy MD. Presumptive
are esculinase-positive and can grow in the identification of group D streptococci: the bile
presence of bile esculin test. Appl Microbiol 1970; 20:245.
4. Lindell SS, Quinn P. Use of bile-esculin agar
• Use a light inoculum when testing E. coli by for rapid differentiation of Enterobacteriaceae.
touching the top of a colony using a J Clin Micro 1975; 1:440.
bacteriological needle and inoculating the 5. Edberg SC, Pittman S, Singer JM. Esculin
agar surface hydrolysis by Enterobacteriaceae. J Clin
Micro 1977; 6:111.
6. MacFaddin JF. Media for isolation,
Quality Control cultivation, identification, maintenance of
bacteria, Vol I. Baltimore: Williams &
After checking for correct pH, colour, depth, Wilkins, 1985.
and sterility, the following organisms are used to 7. Forbes BA, Sahm DF, Weissfeld AS. Bailey
determine the growth performance of the and Scott’s diagnostic microbiology. 10th ed.
completed medium. St. Louis: Mosby, 1998.
8. Murray PR, Baron EJ, Pfaller MA, Tenover
FC, Yolken RH. Manual of clinical
microbiology. 7th ed. Washington D.C.: ASM,
1999.
Original: May 2001

Revised / Reviewed: October 2014

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BILE ESCULIN AGAR

- For in vitro use only - Catalogue No. PB65 & TB65

Our Bile Esculin Agar (BEA) is used as a Recommended Procedure

• This is only a presumptive test for enterococci

Original: May 2001

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