anaeroGRO™

Bacteroides Bile Esculin (BBE) Agar

Cat. no. AG051 Bacteroides Bile Esculin Agar*, Monoplate 1 plate/pouch
Cat. no. AG061 BBE/LKV* Biplate 1 plate/pouch
Cat. no. AG302 DuoPak A, BRU with Hemin and Vitamin K*, Monoplate;
BBE/LKV*, Biplate
2 plates/pouch
Cat. no. AG312 DuoPak B, BRU with Hemin and Vitamin K*, Monoplate;
BBE/PEA*, Biplate
2 plates/pouch
Cat. no. AG303 MultiPak A, BRU with Hemin and Vitamin K*, Monoplate;
PEA*, Monoplate; BBE/LKV*, Biplate
3 plates/pouch

* All AnaeroGRO™ plated media is provided in standard 15x100mm monoplates or biplates. Each plate or set of plates is packaged in an oxygen-free gas flushed foil pouch containing a desiccant and an oxygen scavenger sachet.

INTENDED USE

Hardy Diagnostics AnaeroGRO™ Bacteroides Bile Esculin Agar is an enriched, selective, and differential medium recommended for the isolation and presumptive identification of obligately anaerobic gram-negative bacilli of the Bacteroides fragilis group and Bilophila spp.

SUMMARY

Livingston et al. developed Bacteroides Bile Esculin Agar to accelerate recognition of the Bacteroides fragilis group within 48 hours.(2,9) The medium was developed by combining the components of 20 percent bile stimulation, esculin hydrolysis, catalase production, and kanamycin inhibition tests. Gentamicin was later substituted for kanamycin because it does not lose potency at incubation temperatures.(9)

BBE Agar is useful for rapid isolation and presumptive identification of the B. fragilis group. The medium contains 100ug/ml of gentamicin to inhibit most aerobic organisms and 20% bile to inhibit most other anaerobes. As differential agents, esculin and iron have been incorporated into the agar to aid in detecting the esculin-positive B. fragilis group organisms. BBE Agar is also a useful medium for Bilophila species, which usually produce black-centered colonies on this medium due to H2S production. Other organisms that may grow on this medium are occasional strains of Fusobacterium mortiferum and F. varium, gentamicin-resistant Enterobacteriaceae, enterococci, pseudomonads, staphylococci, and yeast. However, the colony size of the facultative anaerobic organisms is usually less than 1mm in diameter.

AnaeroGRO™ Bacteroides Bile Esculin Agar is packaged in an oxygen-free, reduced state to prevent the formation of toxic oxidized by-products that may damage obligate anaerobes and inhibit the growth of more fastidious species. Culture media that is exposed to environmental oxygen leads to a build-up of reactive oxygen species (ROS) that initiate damaging free radical reactions, which inhibit the growth of anaerobic bacteria. Therefore, ingredients have been added to the AnaeroGRO™ media to neutralize the growth inhibiting effects of peroxide and other reactive oxygen species (ROS) that may develop during the medium's brief exposure to oxygen after it is sterilized and before it is packaged in an oxygen-free environment.

FORMULA

Ingredients per liter of deionized water:*

Tryptic Soy Agar 45.0gm
Oxbile (Oxgall) 20.0gm
Reducing Agents/Peroxide Inhibitors 1.5gm
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 15-30ºC. 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.

The plates must be inoculated immediately after opening the AnaeroGRO™ pouch. After inoculation, the plates must be placed immediately into an anaerobic atmosphere (pouch, jar, or chamber) to ensure optimal growth of anaerobic bacteria.

PRECAUTIONS

PROCEDURE

Specimen Collection: To ensure optimal recovery of anaerobes, specimens should be protected from air (oxygen) as much as possible during collection, transport, and processing. Consult listed references for instructions concerning collection and transport of anaerobes.(1,5,6,7,10)

Method of Use: Open the AnaeroGRO™ pouch just prior to use. The agar surface should be dry prior to inoculation. Minimize specimen exposure to ambient oxygen levels in air. Inoculate and streak the medium to obtain isolated colonies. Incubate in an anaerobic atmosphere at 35-37ºC. for 18-48 hours. Regardless of atmospheric system used, it is important to confirm anaerobiosis by using an anaerobic indicator, such as resazurin (Cat. no. BR55). Observe for growth and blackening of the medium.

Aerotolerance Testing: Confirmation of obligate anaerobic microorganisms should be performed. A Chocolate Agar plate (Cat. no. E14) incubated in 5-10% CO2 is required for aerotolerance testing to detect isolates that require CO2, especially slow-growing, fastidious, facultative or microaerophilic species that do not grow alone on media containing blood (such as Haemophilus and Actinobacillus spp.). Use of traditional blood agar media alone for CO2 incubation may yield false-negative results. An additional Blood Agar plate (Cat. no. A10) incubated in air will further detail the atmospheric requirements and hemolytic properties of facultatively anaerobic microorganisms.

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.(1,3-8,10)

Refer to the Wadsworth-KTL Anaerobic Bacteriology Manual or other texts for more information on the identification of anaerobes.(7)

LIMITATIONS

The plates must be inoculated immediately after opening the AnaeroGRO™ pouch. After inoculation, the plates must be placed immediately into an anaerobic atmosphere (pouch, jar, or chamber) to ensure optimal growth of anaerobic bacteria.

Some strains of B. vulgatus, a member of the B. fragilis group, may 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, members of the B. fragilis group are two to three millimeters in size; whereas, the previously mentioned organisms are less than one millimeter in diameter.(4,8)

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

Some organisms that should grow on AnaeroGRO™ BBE Agar may be inhibited such as occasional strains of Fusobacterium mortiferum and F. varium, and gentamicin-resistant strains of Enterobacteriaceae, enterococci, pseudomonads, staphylococci, and yeast. Consequently, it is recommended that a non-selective medium, such as AnaeroGRO™ Brucella Agar with Hemin and Vitamin K (Cat. no. AG301), be inoculated in parallel to ensure growth of all species present.

Failure to cultivate and/or isolate obligate anaerobes may be due to the following:

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, incubators incinerators, anaerobic culture materials, such as gas generators (Cat. no. AN25US), chambers, transports (Cat. no. S120D), jars (Cat. no. 16000), and oxygen indicators (Cat. no. BR55), 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

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

USER QUALITY CONTROL

Physical Appearance

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

B. fragilis growing on AnaeroGRO™ Bacteriodes Bile Esculin Agar

Bacteroides fragilis (ATCC® 25285) colonies growing on AnaeroGRO™ Bacteriodes Bile Esculin Agar (Cat. no. AG051). Incubated anaerobically for 24 hours at 35ºC.

P. mirabilis inhibited on AnaeroGRO™ Bacteriodes Bile Esculin Agar

Proteus mirabilis (ATCC® 12453) growth inhibited on AnaeroGRO™ Bacteriodes Bile Esculin Agar (Cat. no. AG051). 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. Dowell, V.R., Jr. and T.M. Hawkins. 1987. Laboratory Methods in Anaerobic Bacteriology. In: CDC Laboratory Manual. DHEW Publication No. (CDC) 87-8272. U.S. Department of Health, Education and Welfare, Public Health Service. Center for Disease Control, Atlanta, GA.

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

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

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

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

7. Jousimies-Somer, H.R., S.P. Citron, D. Baron, E.J. Wexler, and H.M. Finegold. 2002. Wadsworth-KTL Anaerobic Bacteriology Manual, 6th ed. Star Publishing Company, New York, N.Y.

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

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

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

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

083016gr