Bacteroides Bile Esculin Agar (BBE) is an enriched selective and differential medium for the isolation and presumptive identification of obligately anaerobic gram-negative bacilli of the Bacteroides fragilis group. Bacteroides Bile Esculin Agar contains gentamicin at a concentration which inhibits most facultative anaerobes. This medium also contains bile, which inhibits anaerobic gram-negative rods except the B. fragilis group and other bile resistant Bacteroides and Fusobacteria. Esculin in the medium permits recognition of esculin hydrolysis by organisms that produce a brown to black coloration in the medium around the colonies. Hemin is added as a growth factor. This medium is prepared, stored and dispensed under oxygen-free conditions to prevent the formation of oxidized products prior to use.

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Formula

Pancreatic Digests of Casein, 15.0 g

Soy Peptone, 5.0 g

Yeast Extract, 5.0 g

Sodium Chloride, 5.0 g

Agar, 15.0 g

Oxgall, 20.0 g

Hemin (0.1% soln), 10.0 ml

Esculin, 1.0 g

Ferric Ammonium Citrate,  0.5 g

Gentamicin, 100.0 mg

Distilled Water,  1000.0 ml

 

Final pH 7.1 +/- 0.4 at 25 degrees C.

Final weight 16.0 g +/- 1.6 for Mono plates.

Final weight 8.0 g +/- 0.8 for Bi-plates.

Precautions

For IN VITRO DIAGNOSTIC USE only.  Approved biohazard precautions and aseptic techniques should be observed when using this product.  This product is for use only by properly-trained and qualified personnel.  Sterilize all biohazard waste prior to disposal.

Storage and Shelf Life

Storage:  Upon receipt, store at room temperature (13°C - 27°C) in original container until used.  Avoid overheating or freezing.  Do not use medium if there are signs of deterioration (shrinking, cracking or discoloration due to oxidation of media) or contamination.  The expiration date applies to the product in its original packaging and stored as directed.  Do not use product past the expiration date shown on the container. 

Shelf Life:  90 days from date of manufacture.

 

Procedure

Specimen Collection:  Specimens for anaerobic culture should be protected from air (oxygen) during collection, transport and processing.  Consult appropriate references for detailed instructions concerning collection and transport of anaerobes.

Methods for Use:  BBE should be inoculated directly with clinical material or a broth that has been previously inoculated from clinical material.  Inoculated plates should be streaked to obtain isolated colonies, immediately placed in an anaerobic atmosphere and incubated at 35-37^oC for 18-48 hours.  Additional periods of incubation may result in loss of selectivity by the medium, resulting in the overgrowth of organisms that should be inhibited.  Additional periods of incubation may be necessary to recover some anaerobes.  Detailed instructions for processing anaerobic cultures can be found in the appropriate references.

Materials Required But Not Provided

Standard microbiological supplies and equipment such as loops, saline blanks, slides, staining supplies, microscope, incinerator / autoclave, incubators, anaerobic chamber / anaerobic jars, disinfectant, other culture media and serological and biochemical reagents.

Limitations

Bacteroides Bile Esculin Agar will not provide complete information for identification of bacterial isolates.  Additional test procedures and media are required for complete identification.  Some organisms that would normally grow on the Bacteroides Bile Esculin Agar medium may be inhibited.  It is recommended that a non-selective medium, such as Anaerobic Brucella Blood Agar also be inoculated from clinical specimens to assure growth of all species present.  Some strains of facultative organisms, which should be inhibited, may grow on Bacteroides Bile Esculin Agar.  A test for aerotolerance should be used to confirm that each colony type is an obligate anaerobe.  Consult reference materials for additional information.

Quality Control

Bacteroides Bile Esculin Agar, if used properly, should support good growth of members of the Bacteroides fragilis group (B. fragilis, B. distasonis, B. thetaiotaomicron, B. ovatus & B. vulgatus) and inhibit growth of most facultative anaerobes and most other non-Bacteroides obligately anaerobic bacteria.  Members of the B. fragilis group should grow as brown to black colonies surrounded by a brown zone in the medium and, with the exception of B. ovatus and B. vulgatus, should produce catalase.

The following organisms are routinely used for quality control performance testing at Anaerobe Systems.

 

User Quality Control: The final determination to the extent and quantity of user laboratory quality control must be determined by the end user. 

If sterility testing is to be performed on this product, the appropriate percentage of the original shipment amount should be incubated anaerobically and aerobically for 48 – 96 hours. 

If the nutritive/inhibitory capacity of this media is to be tested for performance, it is recommended that the following ATCC organisms be evaluated for growth/inhibition.

 

 

Physical Appearance: Bacteroides Bile Esculin Agar should appear translucent green yellow in color.

 

References

1.        Dowell, V. R., Jr. and T. M. Hawkins. 1974. Laboratory Methods in Anaerobic Bacteriology. CDC Laboratory Manual. USDHEW C. D. C. Atlanta, GA 30333.

 

2.        Dowell, V. R., Jr. and G. L. Lombard.  1977.  Presumptive Identification of Anaerobic Non-sporeforming
Gram-negative Bacilli.  USDHEW, CDC.  Atlanta, GA 30333.

 

3.        Dowell, V. R., Jr., G. L. Lombard, F. S. Thompson and A. Y. Armfiedl.  1977.  Media for the Isolation, Characterization, and Identification of Obligately Anaerobic Bacteria.  USDHEW, CDC, Atlanta, GA 30333.

 

4.        Holdeman, L. V., F. P. Cato and W. E. C. Moore.  1977.  Anaerobe Laboratory Manual.  Virginia Polytechnic Institute and State University.  Blacksburg, VA 24061.

 

5.        Somer-Jousimies, H. R., Summanen, P., Citron, D. M., Baron, E. J., Wexler, H. M. and S. M. Finegold.  2002.  Wadsworth – KTL Anaerobic Bacteriology Manual.  Star Publishing Co., Belmont, CA 94002.

 

6.        NCCLS.  Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard - Third Edition.  (2004).  NCCLS document M22-A3.  NCCLS, 940 West Valley Road, Suite 1400, Wayne, PA 19087-1898.