Bile Esculin Agar

BILE ESCULIN AGAR

INTENDED USE REMEL’s Bile Esculin Agar is a solid medium recommended for use in qualitative procedures for the presumptive identification of group D streptococci and enterococci.

SUMMARY AND EXPLANATION Rochaix first demonstrated the value of esculin hydrolysis in the identification of enterococci.1 Meyer and Schonfeld found that 61 of 62 strains of enterococci hydrolyzed esculin in a bile containing medium.2 Facklam and Moody tested 700 strains of streptococci and enterococci representing all known serological groups on a bile esculin medium developed by Swan and determined that 100% of the group D streptococci and enterococci were bile esculin positive.3,4

PRINCIPLE When an organism growing on this medium hydrolyzes the esculin present, esculetin and dextrose are formed. Esculetin reacts with an iron salt to form a dark brown or black complex. Ferric ammonium citrate is incorporated in the medium as the indicator of esculin hydrolysis. The 4% concentration of oxgall (equivalent to 40% bile) in the medium inhibits most strains of streptococci and enterococci other than group D.

REAGENTS (CLASSICAL FORMULA)* Gelatin Peptone ...... 5.0 g Esculin...... 1.0 g Beef Extract...... 3.0 g Ferric Ammonium Citrate ...... 0.5 g Oxgall ...... 40.0 g Agar...... 15.0 g Demineralized Water...... 1000.0 ml pH 6.8 +/- 0.2 @ 25°C

*Adjusted as required to meet performance standards.

PREPARATION OF DEHYDRATED CULTURE MEDIUM 1. Suspend 64 g of medium in 1000 ml of demineralized water. 2. Heat to boiling with agitation to completely dissolve. 3. Sterilize by autoclaving at 121°C for 15 minutes.

PROCEDURE 1. Inoculate the medium with two or three colonies from a pure culture or inoculate from a 24-hour, pure culture in Todd-Hewitt broth using two drops of broth. 2. Incubate aerobically at 35-37°C for up to 72 hours. 3. Observe periodically for blackening of the medium.

INTERPRETATION OF THE TEST Esculin Hydrolysis Positive Test - dark brown to black color diffused into the medium Negative Test - no blackening of the medium

QUALITY CONTROL All lot numbers of Bile Esculin Agar have been tested using the following quality control organisms and have been found to be acceptable. This quality assurance testing conforms with or exceeds NCCLS standards.5 Testing of control organisms should be performed in accordance with established laboratory quality control procedures. If aberrant quality control results are noted, patient results should not be reported.

CONTROL INCUBATION RESULTS *Enterococcus faecalis ATCC 29212 Aerobic, 18-24 h @ 35°C Growth, blackening around colonies Streptococcus bovis ATCC 9809 Aerobic, 18-24 h @ 35°C Growth, blackening around colonies *Streptococcus pyogenes ATCC 19615 Aerobic, 18-24 h @ 35°C Inhibition (partial to complete)

*NCCLS recommended organism

LIMITATIONS Bile Esculin Agar does not contain azide; therefore, gram-negative bacilli may grow on this medium and hydrolyze esculin.

BIBLIOGRAPHY 1. Rochaix, A. 1924. Cr. Soc. Biol. 90:771-772. 2. Meyer, K. and H. Schonfeld. 1926. Zentralbl. Bakteriol. Abt. I. Orig. 99:402-419. 3. Facklam, R.R. and M.D. Moody. 1969. Bact. Proc. M33. 4. Swan, A. 1954. J. Clin Pathol. 7:160-163. 5. National Committee for Clinical Laboratory Standards.1996. Quality Assurance for Commercially Prepared Microbiological Culture Media. 2nd ed. Approved Standard, M22-A2. NCCLS, Wayne, PA.

Refer to the front of the manual for General Information regarding precautions, product storage and deterioration, specimen collection, storage and transportation, materials required, quality control, and limitations.

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

IFU 1190, Revised November 20, 2003 Printed in U.S.A.

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