Brucella blood agar (commonly referred to as BBA (or BRU) is an agar that can be that is used to isolate, quantify as well as partial identification of anaerobes that are fastidious. The agar was initially created to isolate Brucella spp. from potentially hazardous materials, however it has been proven to be effective in the clinical study of strict anaerobes. This agar is often enhanced by Hemin as well as Vitamin K1.
The media contains casein enzyme hydrolysate, peptide digest from animal tissues, as well as yeast extract as the main source of nitrogen, carbon and growth-related nutrients. The sodium bisulfite reduces the redox potential down to an appropriate level for anaerobes. Sheep blood is a source of additional nutrients and permits the identification of hemolytic and non-hemolytic organisms by detecting hemolytic reactions. The sodium chloride in the blood is a source of electrolytes essential to the body and helps maintain the equilibrium of osmotic pressure. Hemin as well Vitamin K1 has shown to aid in the development of more strict anaerobes like Bacteroides as well as Clostridium spp. Agar is the liquidifying agent.
Intended Use
Brucella Agar serves as a medium used to cultivate Brucella organisms. In addition to 5percent blood from horses the medium is employed in qualitative techniques to determine the presence and growth of fastidious and nonfastidious microorganisms that are isolated from clinical and nonclinical samples. Brucella Broth is utilized to cultivate Brucella species and also for cultivating and isolating a broad range of nonfastidious and fastidious microorganisms.
Principle of Brucella Agar
Brucella Agar supports the growth of microorganisms that are fastidious due to the presence of peptones dextrose, peptones, along with yeast extract. Peptones provide organic nitrogen. It is a powerful source of B-complex vitamins. Dextrose is used as a source of energy. It acts as a reducer and sodium chloride helps maintain the equilibrium of osmotic pressure. Agar is the agent used to solidify that is found in Brucella Agar.
Composition of Brucella Agar
| Ingredients | Gms/liter |
| Enzymatic Digest of Casein | 10.00 |
| Enzymatic Digest of Animal Tissue | 10.00 |
| Yeast Extract | 2.000 |
| Sodium Chloride | 5.000 |
| Dextrose | 1.000 |
| Sodium Bisulfite | 0.100 |
| Agar | 15.00 |
Final pH: 7.0 ± 0.2 at 25°C
Preparation and Method of Use of Brucella Agar
- Take 43 g of the medium in 1 Liter of purified water.
- Stir frequently and boil for 1 minute until the medium.
- Autoclave at 121°C for 15 minutes.
- Pour into sterile Petri plates.
- Follow standard protocols to get isolate colonies of specimens.
- Because many pathogens need carbon dioxide for the first isolation, it is recommended to incubate plates at 35 +/- 2 degC for 24 to 72 hours in an oxygenated atmosphere that is supplemented with carbon dioxide.
Result Interpretation on Brucella Agar
After incubation, the majority of plates will exhibit an expanse of growth confluent. Because the procedure of streaking is in essence an “dilution” technique, diminishing number of microorganisms can be found on the areas that are streaked. Therefore, at least one of these areas will show distinct colonies from the species within the specimen. Furthermore, the growth rate of each species could be assessed semi-quantitatively on basis of the rate of growth within each of the areas that are streaked.
| Organisms | Growth |
| Brucella melitensis | Luxuriant, transparent, raised, convex, with an entire edge and a smooth, shiny surface. |
| Brucella ovis | Good, convex colonies |
| Streptococcus pyogenes | Good |
| Brucella suis | Luxuriant, transparent, raised, convex, with an entire edge and a smooth, shiny surface. |
| Staphylococcus aureus subsp. aureus | Inhibited |
| Escherichia coli | Inhibited |
Uses of Brucella Agar
- It is employed to cultivate Brucella spp. and other microorganisms with high-quality in a lab setting.
- It’s a universal environment for cultivating Streptococcus oralis, Streptococcus viridans as well as Neisseria meningitidis.
- In addition to the addition of blood Brucella Agar can be used identify the hemolytic reactions of bacterial species.
- It could be used as a starting point to isolate Campylobacter
- It is also suggested by APHA for the isolation of Brucella species from food sources.
- By adding the addition of 5% of horse’s blood, this solution can be used in qualitative techniques to identify and cultivate of fastidious and non-fastidious microorganisms derived from a variety of clinical and nonclinical samples.
Precautions
- Biosafety Level 2 procedures equipment, facilities and containment equipment are required for all activities that involve clinical samples of animal or human that may contain or possibly contain infectious Brucella spp.
- Biosafety Level 3 procedures equipment, containment facilities and equipment are suggested for any manipulations of the pathogen Brucella Spp. and for animal experiments
Storage
Media is sensitive to temperature and light. Keep plates out of the direct light source at 2-8oC. Plates can be used for up to a week if kept in a clean, sterile space. The media is not to be used if there are there are signs of deterioration hemolysis, contamination, or expiration dates have passed.
Quality Control
Following organisms can be commonly utilized for quality control testing in Anaerobe Systems.
| Organism Tested | Results | Time | Special Reaction |
| Bacteroides fragilis* | Growth | 24 hrs | |
| Prevotella melaninogenica* | Growth | 24 – 48 hrs | Pigmentt (tan color) |
| Fusobacterium necrophorum | Growth | 24 hrs | |
| Fusobacterium nucleatum* | Growth | 24 hrs | |
| Clostridium perfringens* | Growth | 24 hrs | Double Zone of β-hemolysis |
| Peptostreptococcus anaerobius* | Growth | 24 hrs | |
| Staphylococcus aureus or Enterococcus faecalis | Growth | 24 hrs | |
| Escherichia coli | Growth | 24 hrs | |
| Proteus mirabilis | Growth | 24 hrs | |
| Propionibacterium acnes or Clostridium difficile | Growth | 24 – 48 hrs24 hrs |
Limitations of Brucella Agar
- They are not designed to be used in the diagnosis of diseases or other human conditions.
- Because of the variety in their nutrition Some strains might be discovered that are unable to grow or do not grow well in this medium.
- All organisms that are presumed to be anaerobic must be confirmed by a test.
References
- https://www.mycrobe.org/blog/2018/6/28/brucella-blood-agar
- https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/211086.pdf
- https://himedialabs.com/TD/M074.pdf
- https://tools.thermofisher.com/content/sfs/manuals/IFU1250.pdf
- https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/152/145/18795dat.pdf
- https://anaerobesystems.com/products/plated-media/brucella-blood-agar-bru/
