Brucella Agar and Broth

Intended Use

Brucella Agar is a culture medium for the cultivation of Brucella organisms. With the addition of 5% horse blood, the medium is used in qualitative procedures for the isolation and cultivation of nonfastidious and fastidious microorganisms from a variety of clinical and nonclinical specimens.

Brucella Broth is used for the cultivation of Brucella species and for the isolation and cultivation of a wide variety of fastidious and nonfastidious microorganisms.

Summary and Explanation

Brucella Agar was developed for the cultivation of Brucella species from diagnostic specimens, such as blood, and from foods and other potentially contaminated material. Brucella Agar with 5% Horse Blood plates are particularly useful for the cultivation of the more fastidious aerobic and anaerobic microorganisms, including streptococci, pneumococci, Listeria, Neisseria meningitidis and Haemophilus influenzae.

Brucella Broth may be used for the isolation and cultivation of a wide variety of microorganisms including nutritionally fastidious specimens.1 This medium is recommended for the cultivation of Brucella species and was recommended as one of several media suitable for use as the liquid medium component of biphasic blood culture bottles.1,2 It is also used to cultivate Campylobacter spp.3

Principles of the Procedure

Brucella Agar and Brucella Broth support the growth of fastidious microorganisms due to their content of peptones, dextrose and yeast extract. The peptones supply organic nitrogen. The yeast extract is a potent source of the B-complex vitamins. Dextrose is utilized as an energy source. Sodium bisulfite is a reducing agent, and sodium chloride maintains the osmotic equilibrium. Agar is the solidifying agent in Brucella Agar.
In Brucella Agar with 5% Horse Blood plates, the horse blood supplies both the X and V factors which are growth requirements for certain organisms; e.g., Haemophilus influenzae.
3 Sheep and human blood are not suitable for this purpose because they contain enzymes that inactivate the nicotinamide adenine dinucleotide (NAD) which is the V factor.4
Defibrinated horse blood may give hemolytic reactions different than sheep blood.5 Some streptococci (e.g., group D) give hemolytic reactions on horse blood but not on sheep blood and may be mistakenly reported as group A. If a hemolytic reaction is obtained, the organism should be tested with a Taxo™ A bacitracin (0.04 unit) disc and it also should be grouped serologically or tested by the fluorescent antibody method.6 Beta-hemolytic streptococci and Haemophilus haemolyticus may be differentiated by performing a Gram stain on a smear prepared from the colony.

User Quality Control

Identity Specifications
Brucella Agar
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       4.3% solution, soluble in purified water upon
                                    boiling. Solution is light to medium, tan to yellow,
                                    clear to slightly hazy, may contain small
                                    amount of sediment.
Prepared Appearance:     Light to medium, tan to yellow, clear to
                                    slightly hazy.
Reaction of 4.3%
Solution at 25°C:            pH 7.0 ± 0.2
Brucella Broth
Dehydrated Appearance: Fine, homogeneous, free of extraneous material.
Solution:                       2.8% solution, soluble in purified water upon
                                   heating. Solution is pale to medium, tan to
                                   yellow, clear to slightly hazy.
Prepared Appearance:    Pale to medium, tan to yellow, clear to slightly hazy.
Reaction of 2.8%
Solution at 25°C:           pH 7.0 ± 0.2
Cultural Response
Brucella Agar
Prepare the medium per label directions without (plain) and with 5% defibrinated horse blood (HB). Inoculate and incubate at 35 ± 2°C for 3 days with 3-5% CO2 (incubate S. aureus without CO2).

*Minimally one strain of Brucella should be used for performance testing. If these strains are not available, verify performance with a known isolate.
Brucella Broth
Prepare the medium per label directions. Inoculate and incubate at 35 ± 2°C for 7 days with 3-5% CO2 (incubate S. pyogenes for 66-72 hours without CO2).

*Minimally one strain of Brucella should be used for performance testing. If these strains are not available, verify performance with a known isolate.

Formula

Brucella Agar
Approximate Formula* Per Liter
Pancreatic Digest of Casein........................................ 10.0 g
Peptic Digest of Animal Tissue.................................... 10.0 g
Dextrose................................................................... 1.0 g
Yeast Extract.............................................................. 2.0 g
Sodium Chloride......................................................... 5.0 g
Sodium Bisulfite......................................................... 0.1 g
Agar........................................................................ 15.0 g
Brucella Broth
Consists of the same ingredients without the agar.
*Adjusted and/or supplemented as required to meet performance criteria.

Precautions7

1. Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical specimens of human or animal origin containing or potentially containing pathogenic Brucella spp.
2. Biosafety Level 3 practices, containment equipment and facilities are recommended for all manipulations of cultures of the pathogenic Brucella spp. and for experimental animal studies.

Directions for Preparation from Dehydrated Product

1. Suspend the powder in 1 L of purified water:
Brucella Agar – 43 g;
Brucella Broth – 28 g. Mix thoroughly.
2. For the agar, heat with frequent agitation and boil for 1 minute to completely dissolve the powder. For the broth, heat slightly, if necessary, to obtain solution.
3. Autoclave at 121°C for 15 minutes.
4. For preparation of blood plates, add 5 to 10% sterile defibrinated blood to sterile agar which has been cooled to 45-50°C.
5. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

Agar (without or with added blood)
Use standard procedures to obtain isolated colonies from specimens.
Since many pathogens require carbon dioxide on primary isolation, incubate plates at 35 ± 2°C for 24-72 hours in anaerobic atmosphere supplemented with carbon dioxide.
Broth
For liquid specimens, use a sterile inoculating loop to transfer a loopful to the broth medium. Swab specimens may be inserted into the broth after the inoculation of plated media.
Incubate tubes for up to 7 days at 35 ± 2°C in an aerobic atmosphere with or without supplementation with carbon dioxide. For the preparation of biphasic blood culture bottles, aseptically add sterile Brucella Broth to a blood culture bottle containing solidified sterile Brucella Agar, with increased agar at a final concentration of 2.5%. The bottles should contain 5-10% CO2 and be vented. Blood cultures should be incubated at 35°C for up to 30 days with subcultures prepared every 4 to 5 days.1,2

Expected Results

Agar (without or with added blood)
After incubation, most plates will show an area of confluent growth. Because the streaking procedure is, in effect, a “dilution” technique, diminishing numbers of microorganisms are deposited on the streaked areas. Consequently, one or more of these areas should exhibit isolated colonies of the organisms contained in the specimen. Further, growth of each organism may be semi-quantitatively scored on the basis of growth in each of the streaked areas.
Broth
Growth in the tubes is indicated by the presence of turbidity compared with an uninoculated control. If growth appears, cultures should be examined by Gram stain and subcultured onto appropriate media; e.g., Trypticas Soy Agar with 5% Sheep Blood and/or Brucella Agar and Chocolate II Agar, Eosin Methylene Blue Agar, Levine or MacConkey II Agar.

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