BRUCELLA AGAR WITH HEMIN AND VITAMIN K (H & K)

Cat. no. A30 Brucella Agar with H & K,15x100mm Plate, 19ml 10 plates/bag
Cat. no. W23 Brucella Agar with H & K,15x100mm Plate, 26ml 10 plates/bag
Cat. no. H05 Brucella Agar with H & K, 15x150mm Plate, 69ml 10 plates/bag
Cat. no. J74 Brucella Agar with H & K / Anaerobic PEA, 15x100mm Biplate,
10ml/10ml
10 plates/bag
Cat. no. J87 Brucella Agar with H & K / LKV Agar, 15x100mm Biplate,
10ml/10ml
10 plates/bag

INTENDED USE

Hardy Diagnostics Brucella Agar with H & K is recommended for use in the primary isolation and cultivation of anaerobic microorganisms.

SUMMARY

Brucella Agar with H & K is modification of the formulation given by the American Society for Microbiology (ASM). (4) According to Finegold, this media is reported preferable to Heart Infusion Blood Agar Base for the cultivation of anaerobic bacteria. (2) Onderdonk, et al., and Weinstein, et al., both reported the addition of Hemin. (7,10) Summanen, et al., described supplementing the medium with vitamin K. (9) The incorporation of these additives, into Brucella Agar with H & K, enhances the cultivation of some species of anaerobes. (6)

Brucella Agar with H & K is a rich nutrient base that supports the growth of fastidious organisms. The media contains dextrose which provides an energy source; peptones to provide nitrogenous compounds, and yeast extract to supply B vitamins. Growth factors required by some anaerobic bacteria are provided for in the added sheep blood, which also allows the demonstration of hemolytic reactions. Hemin and vitamin K are incorporated into the medium to enhance the growth of gram-positive spore-formers and Bacteroides species. (6)

FORMULA

Ingredients per 950ml of deionized water:*

Pancreatic Digest of Casein 10.0gm
Pancreatic Digest of Animal Tissue 10.0gm
Sodium Chloride 5.0gm
Yeast Extract 2.0gm
Dextrose 1.0gm
Sodium Bisulfite 0.1gm
Hemin 10.0mg
Vitamin K 10.0mg
Sheep Blood, Defibrinated 50.0ml
Agar 15.0gm

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), hemolysis, 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: Consult listed references for information on specimen collection. (1-5) 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.

When possible the clinical specimen should be inoculated directly onto the medium to prevent loss of organism viability. A liquid specimen may be directly applied to the agar surface and streaked with a sterile inoculating loop. Specimens for anaerobic culture should be plated on both non-selective and selective media.

Method of Use: Consult listed references for the correct inoculation procedure. (1-5,9) Incubate plates anaerobically at 35-37ºC. for up to 72 hours. Confirmation of anaerobic organisms should be performed by subculturing to an aerobic Blood Agar plate (Cat. no. A10). Examine anaerobic colonies for hemolytic reaction, colony morphology, gram stain, and further biochemical testing.

INTERPRETATION OF RESULTS

Consult listed references for the interpretation of growth of anaerobic species. (1-5,9)

LIMITATIONS

It is suggested that Brucella Agar with H & K plates be reduced, prior to use, for a minimum of 24 hours by placing them in an anaerobe jar at room temperature.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, slides, anaerobe jars or bags, anaerobic generators and indicators, incinerators, microscopes, 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 24-48hr 35°C Anaerobic Growth
Bacteroides levii
ATCC ® 29147
A 24-48hr 35°C Anaerobic Growth
Clostridium perfringens
ATCC ® 13124
A 24-48hr 35°C Anaerobic Growth; beta-hemolysis
Fusobacterium nucleatum
ATCC ® 25586
A 24-48hr 35°C Anaerobic Growth
Peptostreptococcus anaerobius
ATCC ® 27337
A 24-48hr 35°C Anaerobic Growth

User Quality Control

PHYSICAL APPEARANCE

Brucella Agar with H & K should appear opaque, and cherry red in color.

F. nuleatum growing on Brucella Agar with H and K

Fusobacterium nucleatum (ATCC ® 25586) colonies growing on Brucella Agar with H and K (Cat. no. A30). Incubated anaerobically for 24 hours at 35ºC.

Bacteroides fragilis (ATCC ® 25285) colonies growing on Brucella Agar with H and K (Cat. no. A30). Incubated anaerobically for 24 hours at 35ºC.

B. levii growing on Brucella Agar with H and K

Bacteroides levii (ATCC ® 27337) colonies growing on Brucella Agar with H and K (Cat. no. A30) Incubated anaerobically for 24 hours at 35ºC.

C. perfringens growing on Brucella Agar with H and K

Clostridium perfringens (ATCC ® 13124) colonies growing on Brucella Agar with H and K (Cat. no. A30). Incubated anaerobically for 48 hours at 35ºC.

P. anaerobius growing on Brucella Agar with H and K

Peptostreptococcus anaerobius (ATCC ® 27377) colonies growing on Brucella Agar with H and K (Cat. no. A30). Incubated anaerobically for 48 hours at 35ºC.

Brucella Agar with H and K

Uninoculated plate of Brucella Agar with H and K (Cat. no. A30).

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. 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. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

7. Onderdonk, A.B., et al. 1974. Infect. Immun.; 10:1256.

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

9. Summanen, P., et al. 1993. Wadsworth Anaerobic Bacteriology Manual, 5th ed. Star Publishing Company, Belmont, CA.

10. Weinstein, W.M., et al. 1974. Infect. Immun.; 10:1250.


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