Brucella Blood Agar (BRU) is an enriched non-selective agar medium. Brucella Blood Agar is intended for the isolation, quantitation and partial identification of obligate anaerobic bacteria from clinical specimens.  It is supplemented with vitamin K₁ and Hemin to facilitate the recovery and pigment production of Prevotella melaninogenica.  Sheep blood has been added for the observation of hemolytic reactions as seen by the double zone b - hemolysis of Clostridium perfringens.  This medium will also support the growth of aerobic and microaerophilic bacteria if incubated appropriately.  Brucella Blood Agar is also suitable for use in susceptibility testing, antibiotic differential disk examination and spot biochemical testing.  This medium is prepared, stored and dispensed under oxygen-free conditions to prevent the formation of oxidized products prior to use

Formula

Pancreatic Digest of Casein, 10.0 g

Soy Peptone, 3.0 g

Animal Tissue, 10.0 g

Dextrose, 1.0 g

Yeast Extract, 2.0 g

Sodium Chloride, 5.0 g

Sodium Bisulfite, 0.1 g

Tryptophan, 0.2 g

Hemin (0.1% soln), 5.0 ml

Vitamin K₁ (1% soln), 1.0 ml

L-Cystine, 0.4 g

Agar, 15.0 g

Sheep Blood, 45.5 ml

Distilled Water, 1000.0 ml

 

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

Final weight 16.0 g +/- 1.6.

Final weight 68.0 g +/- 3.4 for AS-614

Final depth 4.0 mm +/-0.4 mm for AS-614

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 use.  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: Brucella Blood Agar 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 and immediately placed in an anaerobic atmosphere and incubated at 35-37^oC for 18-48 hours.  Additional periods of incubation may be necessary to recover some anaerobes.  Detailed instructions for processing anaerobic cultures can be found in the appropriate references (1, 2, 3, 4, 5, and 6).

Materials Required But Not Provided

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

Limitations

Brucella Blood Agar will not provide complete information for identification of bacterial isolates.  Additional test procedures and media are required for complete identification.  In some cases, Brucella Blood Agar may be overgrown with swarming Proteus spp. or Clostridium spp.  It is recommended that selective media such as Anaerobic Brucella Laked Blood Agar with Kanamycin and Vancomycin (LKV) and/or Anaerobic Brucella Blood Agar with Phenylethyl Alcohol (PEA) also be inoculated from clinical specimens to prevent such overgrowth and thus provide isolated colonies.  Consult reference materials for additional information.

Quality Control

Brucella Blood Agar should support good growth of obligate anaerobes found in clinical infections.  In addition, this media should support typical pigment production by Prevotella melaninogenica and typical double zone of hemolysis around colonies of Clostridium perfringens. 

The following organisms are routinely used for quality control testing (nutritive capacity) at Anaerobe Systems.

Organism Tested for BRU	ATCC #	Results	Time (Hours)	Special Reaction
Bacteroides fragilis **	25285	Growth	24 h
Prevotella melaninogenica **	25845	Growth	24 – 48 h	Pigmentt (tan color)
Fusobacterium necrophorum	25286	Growth	24 h
Fusobacterium nucleatum **	25586	Growth	24 h
Clostridium perfringens **	13124	Growth	24 h	Double Zone of hemolysis
Peptostreptococcus anaerobius **	27337	Growth	24 h
Staphylococcus aureus   or    Enterococcus faecalis	25923     29212	Growth	24 h
Escherichia coli	25922	Growth	24 h
Proteus mirabilis	12453	Growth	24 h
Propionibacterium acnes   or  Clostridium difficile	6919      9686	Growth	24 – 48 h 24 h

** Organisms specified by NCCLS for Quality Control testing of Anaerobic Blood Agars.

  ^t  Pigment production may require more than 48 hours incubation

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

 

Organism	ATCC	Expected Results	Special Reactions
B. fragilis	25285	24 hrs
P. melaninogenica	29147	24-48 hrs	Pigment t
F. necrophorum	25286	24 hrs
C. perfringens	13124	24 hrs	Double zone of b -hemolysis
P. anaerobius	27337	24 hrs

^t  Pigment production may require more than 48 hours incubation

Physical Appearance:  Brucella Blood Agar should appear opaque burgundy red in color.  For AS-614, the media is in a 150mm x 15mm plate. All other catalog numbers for Brucella Blood Agar come in standard sized Petri dishes.

 

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.        Jousimies-Somer, 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.