Cat. no. G11 Bile Esculin Agar (BEA) with Azide, 15x100mm
Plate, 18ml
10 plates/bag
Cat. no. K11 Bile Esculin Azide Broth, 15x103mm Tube, 5ml 20 or 100 tubes/box
Cat. no. J66 Bile Esculin Agar (BEA) with Azide / Columbia CNA,
15x100mm Biplate, 10ml/10ml
10 plates/bag


Hardy Diagnostics Bile Esculin with Azide Media are recommended for the isolation and differentiation of group D streptococci from non-group D streptococci.


Hardy Diagnostics Bile Esculin Agar with Azide is also referred to as Pfizer Selective Enterococcus (PSE) Agar. Esculin hydrolysis was first described by Rochaix in 1924.(8) Swan first introduced the use of Bile Esculin Agar in 1954.(9) Facklam and Moody, in 1970, determined that the use of the bile esculin test was a reliable way of identifying group D streptococci from non-group D streptococci.(3) When using BEA in biochemical testing of group D streptococci, they found that all group D streptococci will blacken this medium.(3) Other researchers have used BEA for the presumptive identification of Enterobacter, Klebsiella and Serratia spp. only.

This medium contains esculin, ferric citrate to provide ferric ions, and 1% oxbile to inhibit most other strains of non-group D streptococci. Sodium azide is incorporated into the medium to inhibit gram-negative bacteria. Esculin is hydrolyzed by group D streptococci to form dextrose and esculetin. This compound reacts with the ferric ions contained within the medium, turning the medium dark brown to black. Thus, the tolerance to the presence of bile and the hydrolysis of esculin provide the means to presumptively identify group D streptococci.


Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 16.0gm
Oxbile 10.0gm
Yeast Enriched Meat Peptone 9.5gm
Sodium Chloride 5.0gm
Sodium Citrate 1.0gm
Esculin 1.0gm
Ferric Ammonium Citrate 0.5gm
Sodium Azide 0.25gm

In addition, Bile Esculin Agar (BEA) with Azide contains:

Agar 15.0gm

Final pH 7.1 +/- 0.3 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store Bile Esculin Agar with Azide at 2-8ºC. and Bile Esculin Azide Broth at 2-30ºC. Store media away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat and freezing.



Specimen Collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Consult listed references for information on specimen collection.(1-5)

Method of Use: Allow the medium to warm to room temperature prior to inoculation. BEA with Azide is a primary isolation medium. This medium enhances the recovery and isolation of individual colonies from clinical specimens. Inoculate agar and streak for isolation using the four quadrant method. Using a sterile inoculating loop, inoculate broth with well isolated colonies. Incubate inoculated media in an aerobic atmosphere at 35ºC. for 18-48 hours. Observe for growth and blackening of the medium.


A positive test result denoting the presence of group D streptococci is any blackening of the media. A negative test result is no blackening of the media after 48 hours of incubation.

Consult listed references for information pertaining to colony morphology and biochemical tests required for identification.(1,2,4,6)


Some strains of Staphylococcus and Aerococcus can grow in the presence of bile and can hydrolyze esculin.

A heavy inoculum on Bile Esculin Azide Media may cause difficulties in interpreting the bile esculin test. Excess inoculum decreases the ability of the oxbile to inhibit 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. Growth without blackening of this medium does not constitute a positive test for group D streptococci.


Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bile Esculin Agar with Azide and Bile Esculin Azide Broth:
Enterococcus faecalis
ATCC ® 29212
A 24-48hr 35°C Aerobic Growth; blackening of media around colonies or blackening of broth
Escherichia coli
ATCC ® 25922
B 24-48hr 35°C Aerobic Partial inhibition; small colorless colonies. Growth inhibited in broth.
Streptococcus pyogenes
ATCC ® 19615
B 24-48hr 35°C Aerobic Partial to complete inhibition. Growth inhibited in broth.

User Quality Control


E. faecalis growing on Bile Esculin Agar with Azide

Enterococcus faecalis (ATCC ® 29212) colonies growing on Bile Esculin Agar with Azide (Cat. no. G11). Incubated aerobically for 24 hours at 35ºC.

S. pyogenes inhibited on Bile Esculin Agar with Azide

Streptococcus pyogenes (ATCC ® 19615) growth inhibited on Bile Esculin Agar with Azide (Cat. no. G11). Incubated aerobically for 24 hours at 35ºC.


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Facklam, R.R., and M.D. Moody. 1970. Appl. Microbiol.; 20:245.

4. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I & II. American Society for Microbiology, Washington, D.C.

5. McFaddin, J.F. 1985.Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

6. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

7. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

8. Rochaix, Cr. 1924. Soc. Biol., Paris; 90:771.

9. Swan, A. 1954. J. Clin. Path.; 7:160-163.

ATCC is a registered trademark of the American Type Culture Collection.