Brucella Blood Agar Deep (BRU DEEP), is intended for the isolation, quantitation and partial identification of obligate anaerobic bacteria from clinical specimens. This medium will also support the growth of aerobic and microaerophilic bacteria if incubated appropriately. After additional supplementation, it is suitable for use in the agar dilution antibiotic susceptibility testing method. BRU DEEP is an enriched non-selective medium. The basic nutritive properties consist of casein peptones, dextrose and yeast extract. BRU DEEP is further supplemented with vitamin K1 and hemin to facilitate the recovery and pigment production of Prevotella melaninogenica. The addition of 5% per volume of sheep blood will facilitate observation of hemolytic reactions as seen by the double zone b-hemolysis of Clostridium perfringens. This medium is prepared, stored and dispensed under oxygen-free conditions to prevent the formation of oxidized products prior to use.

      Anaerobe Systems PRAS Brucella Agar
             Deep Broth (BRU-DEEP) AS-105

 

          View the full product insert: BRU DEEP Insert.pdf

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

Agar, 15.0 g

Sodium Chloride, 5.0 g

Sodium Bisulfite, 0.1 g

Tryptophan, 0.2 g

L-Cystine, 0.4

Hemin (0.1% soln), 5.0 ml

Vitamin K₁ (1% soln), 1.0 ml

Distilled Water, 1000.0 ml

 

Final pH 7.2 +/- 0.2 at 25 degrees C.

Final volume 18.0 g +/- 1.8.

                                                                                                               

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 packaging 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: 6 months from date of manufacture.

 

 Procedure

Method for Preparation: Melt tubes of agar and cool to 50^oC; at this point add 1.0 ml laked or defibrinated sheep blood.  Mix contents thoroughly by inverting or rotating tubes.  Pour contents into a standard round petri plate and allow to solidify.  After the agar has solidified, place plates in an incubator at 35 – 37^oC for 30 to 45 minutes (with lid slightly ajar) to allow the excess moisture to evaporate.

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:  After following the above method for preparation, BRU DEEP with 5% sheep blood 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, immediately placed in an anaerobic atmosphere and incubated at 35-37^oC for 

18 – 48 hours.  Detailed instructions for processing anaerobic cultures can be found in the appropriate references.

Materials Required But Not Provided

Water bath, defibrinated or laked sheep blood, petri plates, standard microbiological supplies and equipment such as loops, saline blanks, slides, staining supplies, microscope, incinerator / autoclave, incubators, anaerobic chamber or anaerobic jars, other culture media and serological and biochemical reagents.

 

Limitations

BRU DEEP will not provide complete information for identification of bacterial isolates.  Additional test procedures and media are required for complete identification.  In some cases, after inoculation with clinical specimen, BRU DEEP with 5% sheep blood may be overgrown with swarming Proteus spp. or Clostridium spp.  Consult reference materials for additional information.

 

Quality Control

BRU DEEP, when supplemented with 5% sheep blood, should support good growth of obligate anaerobes found in clinical infections.  In addition, this media should support typical pigment production by P. melaninogenica and typical double zone of hemolysis around colonies of C. perfringens.

The following organisms are routinely used for quality control testing at Anaerobe Systems. 

Organism Tested	ATCC #	Results	Time (Hours)	Special Reaction (with addition of blood)
Bacteroides fragilis	25285	Growth	24 h
Prevotella melaninogenica	25845	Growth	24–48 h	Pigment t (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 9689	Growth Growth	24-48 h 24 h

 

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 medium is to be tested for performance, it is recommended that the following ATCC organisms be evaluated for growth.

Organism	Growth in Hours	Expected Results (with addition of blood)
B. fragilis	24 hrs
P. melaninogenica	24-48 hrs	Pigment
F. necrophorum	24 hrs
C. perfringens	24 hrs	Double zone of b-hemolysis
P. anaerobius	24 hrs

 

Physical Appearance:  BRU DEEP should appear opaque and tan in color in a 20mm x 113mm glass tube.

 

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., G. L. Lombard, S. F. Thompson and A. Y. Armfield.  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.