Cat. no. L29 Esculin Agar, 16x100mm Tube, 6.5ml Slant 20 or 100 tubes/box


Hardy Diagnostics Esculin Agar is recommended for the detection of esculin hydrolysis by microorganisms. It is particularly useful in the differentiation of Bacteroides species.


In 1909, Harrison and Van der Leck observed the ability of organisms to hydrolyze esculin. (6) The esculin hydrolysis test was used in 1924 by Rochaix for use in the identification of group D streptococci. (7) Smith, and later Dowell, employed its use in the differentiation of anaerobic bacteria and Bacteroides spp., respectively. (8,9)

Esculin Agar consists of a heart infusion basal medium supplemented with vitamin K, hemin, 0.1% esculin and ferric citrate. The nutritional growth requirements of bacteria are supplied by the heart infusion. Vitamin K and hemin are added to enhance the growth of anaerobic microorganisms. Esculin and ferric citrate allow for the detection of esculin hydrolyzing microorganisms.

Esculin, a water soluble glycoside, is hydrolyzed by certain bacteria to yield glucose and esculetin. Esculetin reacts with the ferric ions to produce a black colored complex that surrounds the colonies. Microorganisms that are not capable of hydrolyzing esculin will not produce a blackening in the medium.


Ingredients per liter of deionized water:*

Dipeptone 10.0gm
Pancreatic Digest of Casein 6.0gm
Sodium Chloride 5.0gm
Beef Heart Infusion 2.0gm
Yeast Extract 2.0gm
Esculin 1.0gm
Ferric Citrate 0.5gm
Vitamin K 10.0mg
Hemin 5.0mg
Agar 15.0gm

Final pH 7.0 +/- 0.2 at 25ºC.

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


Storage: Upon receipt store at 2-30ºC. 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, moisture, and freezing.



Specimen Collection: Specimen collection is not applicable since this medium is not intended for primary isolation. As a general rule, 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:

1. Using a fishtail motion, inoculate the surface of the slant with 3-4 pure colonies that are 18-24 hours old.

2. Incubate the inoculated medium aerobically or anaerobically (dependant upon the isolates growth requirements) at 35-37ºC. for 24-72 hours.

3. Observe for the development of a brownish-black to black color in the medium surrounding the colonies.


A positive esculin hydrolysis test is indicated by the development of a brownish-black to black color in the medium surrounding the colonies.

A negative esculin hydrolysis test is denoted by no color change in the medium.


Iron sulfide, which appears as a sooty black color on the medium, may be produced, and thus, may lead to difficulty in test interpretation.


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
Enterococcus faecalis
ATCC ® 29212
A 24-48hr 35°C Aerobic Growth; blackening around colonies
Escherichia coli
ATCC ® 25922
B 24-48hr 35°C Aerobic Growth; no color development in media

User Quality Control


Esculin Agar should appear clear, and medium amber with a slight blue tinge in color.

E. faecalis growing on Esculin Agar

Enterococcus faecalis (ATCC ® 29212) colonies growing on Esculin Agar (Cat. no. L29). Incubated aerobically for 24 hours at 35ºC.

E. coli growing on Esculin Agar

Escherichia coli (ATCC ® 25922) colonies growing on Esculin Agar (Cat. no. L29). Incubated aerobically for 24 hours at 35ºC.

Esculin Agar

Uninoculated tube of Esculin Agar (Cat. no. L29).


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

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

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

5. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

6. Harrison, R.C., and Van der Leck, J. 1909. Esculin Bile Salt Media for Water Analysis, Bakt. Abt.; 222:549.

7. Rochaix, A. 1924. Cr. Soc. Biol., Paris, France; 90:771.

8. Smith, L. 1975. The Pathogenic Anaerobic Bacteria, 2nd ed. Springfield, IL.

9. D.H.E.W. Publ., CDC; 72:8272, 1974.

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