BACTEROIDES BILE ESCULIN (BBE) AGAR

Cat. no. G05 BBE Agar, 15x100mm Plate, 18ml 10 plates/bag
Cat. no. J102 BBE / LKV, 15x100mm Biplate, 10ml/10ml 10 plates/bag

INTENDED USE

Hardy Diagnostics Bacteroides Bile Esculin Agar (BBE) is recommended for use in the rapid isolation and presumptive identification of Bacteroides fragilis group.

SUMMARY

Livingston, et al., developed Bacteroides Bile Esculin Agar to accelerate the recognition of Bacteroides fragilis group by providing tentative identification from a primary plate medium within 48 hours.(8,11) The selective and differential media was developed by combining the components of twenty percent bile stimulation, esculin hydrolysis, catalase production, and kanamycin inhibition tests. Later, gentamicin was substituted for kanamycin. Gentamicin proved to be an effective substitute, as it does not lose its activity at incubation temperatures and can be incorporated into BBE Agar before autoclaving.(11)

The basal medium is composed of Tryptic Soy Agar (TSA) and is supplemented with the following: twenty percent bile (oxgall) to stimulate growth of B. fragilis group while inhibiting other anaerobes; esculin and ferric ammonium citrate to detect esculin hydrolysis; hemin which serves as a growth factor and allows testing for catalase production; and gentamicin which inhibits most facultative anaerobes.

B. fragilis group hydrolyze esculin to form dextrose and esculetin. This compound reacts with the ferric ions contained within the medium, turning the medium around the colonies a dark brown to black color. Thus, the tolerance to the bile and hydrolysis of esculin provide the means to presumptively identify B. fragilis group.

FORMULA

Ingredients per liter of deionized water:*

Tryptic Soy Agar 45.0gm
Oxbile (Oxgall) 20.0gm
Esculin 1.0gm
Ferric Ammonium Citrate 0.5gm
Hemin 12.0mg
Gentamicin 100.0mg

Final pH 7.0 +/- 0.3 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. 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.

PRECAUTIONS

PROCEDURE

Specimen Collection: To assure the recovery of anaerobes, specimens should be protected from air (oxygen) during collection, transport, and processing. Consult listed references for instructions concerning collection and transport of anaerobes.(1,2,4,6)

Method of Use: The medium should be warmed to room temperature and agar surface should be dry, prior to inoculation. Inoculate and streak the medium to obtain growth of isolated colonies. Incubate in an anaerobic atmosphere at 35-37ºC. for 18-48 hours. Observe for growth and blackening of the medium.

INTERPRETATION OF RESULTS

This product is used in conjunction with other biochemical tests to identify cultures of isolated organism. Esculin hydrolysis is indicated by a browning or blackening in the medium surrounding a colony.

Consult listed references for the identification of colony morphology and further biochemical tests required for identification.(1,2.4,6)

LIMITATIONS

Some strains of B. vulgatus, a member of B. fragilis group, can test esculin-negative.

Esculin can be hydrolyzed by some bile-resistant, non- B. fragilis group microorganisms. Some examples of such microorganisms are Bacteroides splanchnicus, Fusobacterium mortiferum, Klebsiella pneumoniae, Enterococcus species, and yeasts. In general, B. fragilis group are two to three millimeters in size, whereas, the previously mentioned organisms are less than one millimeter in diameter.(9,10)

It is suggested that BBE Agar plates be reduced, prior to use, by placing them in an anaerobic atmosphere at room temperature for a period of 6-24 hours.

Many anaerobes are sensitive to oxygen during the log phase of growth and may be killed by exposure to oxygen before the colonies are fully developed. It may be necessary, therefore, to incubate an inoculated culture for 48 hours (three to five days is preferable) before exposing the culture to room air.

Some organisms which should grow on BBE Agar may be inhibited. It is recommended that a non-selective medium such as Brucella with H & K (Cat. no. A30) be inoculated in parallel in order to assure growth of all species present.

MATERIALS REQUIRED BUT NOT PROVIDED

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.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Bacteroides fragilis
ATCC® 25285
A 18-24hr 35°C Anaerobic Growth; blackening of the media
Proteus mirabilis
ATCC® 12453
B 18-24hr 35°C Aerobic Inhibited

User Quality Control

PHYSICAL APPEARANCE

Bacteroides Bile Esculin (BBE) Agar should appear opalescent, and medium amber with blue tinge in color.

B. fragilis growing on Bacteriodes Bile Esculin Agar

Bacteroides fragilis (ATCC® 25285) colonies growing on Bacteriodes Bile Esculin Agar (Cat. no. G05). Incubated anaerobically for 24 hours at 35ºC.

P. mirabilis inhibited on Bacteriodes Bile Esculin Agar

Proteus mirabilis (ATCC® 12453) growth inhibited on Bacteriodes Bile Esculin Agar (Cat. no. G05). Incubated aerobically for 24 hours at 35ºC.

REFERENCES

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. MacFaddin, 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-A2. Clinical Laboratory Standards Institute (CLSI - formerly NCCLS), Villanova, PA.

8. Dowell, V.R., Jr., et al. 1974. Laboratory Methods in Anaerobic Bacteriology, CDC Lab. Manual, USDHEW, Washington, D.C.

9. Finegold, S.M. and E.J. Baron. 1986. Bailey and Scott's Diagnostic Microbiology, 7th ed. C.V. Mosby, St. Louis, MO.

10. Lennette, E.H., et al. 1985. Manual of Clinical Microbiology, 4th ed. American Society for Microbiology Washington, D.C.

11. Livingston, S.J., et al. 1978. J. Clin. Microbiol.; 7:448-453.


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121415hh