anaeroGRO™

Brucella Agar with Hemin and Vitamin K (BRU)

Cat. no. AG301 Brucella Agar with Hemin and Vitamin K*, Monoplate 1 plate/pouch
Cat. no. AG304 Brucella Agar with Hemin and Vitamin K* 4Pak, Monoplate 4 plates/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
Cat. no. AG313 MultiPak B, BRU with Hemin and Vitamin K*, Monoplate;
LKV*, Monoplate; PEA*, Monoplate
3 plates/pouch

* These AnaeroGRO™ plated media are 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™ Brucella Agar with Hemin and Vitamin K is recommended for use in the primary isolation, quantitation, and partial identification of obligately anaerobic microorganisms from clinical specimens. This non-selective medium is also suitable for the growth of aerobic and microaerophilic bacteria when incubated under the appropriate conditions.

SUMMARY

Brucella Agar with Hemin and Vitamin K is a modification of the formulation given by the American Society for Microbiology (ASM).(4) According to Finegold, this medium is preferable to Heart Infusion Blood Agar Base for the cultivation of anaerobic bacteria.(8) Onderdonk et al. and Weinstein et al. both reported the addition of hemin.(9,11) Jousimies-Somer et al. further describe supplementing the medium with vitamin K.(5) It has been shown that Brucella Agar supports the growth of anaerobic gram-negative bacilli better than CDC (trypticase soy) or Schaedler's agar.(5)

AnaeroGRO™ Brucella Agar with Hemin and Vitamin K is an enriched medium designed to support and enhance the growth of fastidious microorganisms. The medium contains dextrose for energy, peptones to provide nitrogenous compounds, and yeast extract as a source of B vitamins for cell maintenance and metabolism. Sheep blood provides additional growth factors required by some fastidious microorganisms and can also be used to assess hemolytic reactions as seen by the double zone beta-hemolysis of Clostridium perfringens. Hemin and vitamin K are nutritious supplements known to enhance the cultivation of some species of anaerobes, and to further promote the pigment production of Prevotella melaninogenica.(7) The medium is also suitable for use in susceptibility testing, differential disk, and spot biochemical testing.

AnaeroGRO™ Brucella Agar with Hemin and Vitamin K 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:*

Pancreatic Digest of Casein 10.0gm
Pancreatic Digest of Animal Tissue 10.0gm
Sodium Chloride 5.0gm
Yeast Extract 2.0gm
Reducing Agents/Peroxide Inhibitors 1.5gm
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 15-30ºC. 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.(1-6,8) Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat, cold and oxygen exposure. If there is to be a delay in processing, the specimen should be inoculated into an appropriate transport medium (Cat. no. S120D) and refrigerated until inoculation.

When possible, clinical specimens should be inoculated directly onto the medium to prevent loss of organism viability. Minimize specimen exposure to ambient oxygen levels in air.

Method of Use: Consult listed references for the correct inoculation procedure.(1-6,8) Open the AnaeroGRO™ pouch just prior to use and immediately apply a liquid specimen directly to the agar surface; streak with a sterile inoculating loop to obtain isolated colonies. An enrichment broth, such as AnaeroGRO™ Thioglycollate Broth with Hemin and Vitamin K (Cat. no. AG22H), should be inoculated concurrently with primary isolation plates. For best results, specimens should be plated onto non-selective and selective media.

Immediately after culture, incubate plates anaerobically at 35-37ºC. for up to 72 hours. Some fastidious anaerobes may require additional periods of incubation for proper recovery. Regardless of atmospheric system used, it is important to confirm anaerobiosis by using an anaerobic indicator, such as resazurin (Cat. no. BR55).

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

Consult listed references for the interpretation of growth of anaerobic species.(1-6,8)

Examine colonies using a dissecting microscope and a long-wave UV lamp to determine fluorescence. Certain strains of pigment producing Bacteroides spp. should fluoresce orange to brick-red under long-wave UV light. Fluorescence should be visible prior to colony pigmentation.

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

Refer to the Wadsworth-KTL Anaerobic Bacteriology Manual or other texts for more information on the identification of anaerobes.(5)

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.

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. Identification disks that are useful on Brucella with H and K would include Vancomycin (Cat. no. Z7501, Kanamycin (Cat. no. Z7191), SPS (Cat. no. Z7381), Colistin (Cat. no. Z78411) and Nitrate (Cat. no. Z7071).

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

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

USER QUALITY CONTROL

Physical Appearance

AnaeroGRO™ Brucella Agar with Hemin and Vitamin K should appear opaque, and medium to dark red in color.

F. nuleatum growing on AnaeroGRO™ Brucella Agar with H and K

Fusobacterium nucleatum (ATCC® 25586) colonies growing on AnaeroGRO™ Brucella Agar with H and K (Cat. no. AG301). Incubated anaerobically for 24 hours at 35ºC.

B. fragilis growing on AnaeroGRO™ Brucella Agar with H and K

Bacteroides fragilis (ATCC® 25285) colonies growing on AnaeroGRO™ Brucella Agar with H and K (Cat. no. AG301). Incubated anaerobically for 24 hours at 35ºC.

B. levii growing on AnaeroGRO™ Brucella Agar with H and K

Bacteroides levii (ATCC® 29147) colonies growing on AnaeroGRO™ Brucella Agar with H and K (Cat. no. AG301). Incubated anaerobically for 24 hours at 35ºC.

C. perfringens growing on AnaeroGRO™ Brucella Agar with H and K

Clostridium perfringens (ATCC® 13124) colonies growing on AnaeroGRO™ Brucella Agar with H and K (Cat. no. AG301). Incubated anaerobically for 48 hours at 35ºC.

P. anaerobius growing on AnaeroGRO™ Brucella Agar with H and K

Peptostreptococcus anaerobius (ATCC® 27377) colonies growing on AnaeroGRO™ Brucella Agar with H and K (Cat. no. AG301). Incubated anaerobically for 48 hours at 35ºC.

AnaeroGRO™ Brucella Agar with H and K

Uninoculated plate of AnaeroGRO™ Brucella Agar with H and K (Cat. no. AG301).

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. 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. 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.

6. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

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

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

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

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

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


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

083016gr