Brucella Agar – Composition, Principle, Preparation, Results, Uses

What is Brucella Agar?

• Brucella Agar is a specialized medium used for the isolation and cultivation of Brucella species, which are intracellular parasites that can cause epizootic abortions in animals and septicemic febrile illness or localized infections in humans. These bacteria are highly fastidious, meaning they have specific nutritional requirements and therefore need a nutrient-rich medium to grow successfully.
• The Brucella Agar Base, when supplemented with Campylobacter Supplements, can also be used for the isolation of Campylobacter, another group of bacteria. This makes it a versatile medium for the isolation and cultivation of both Brucella and Campylobacter species from various sources.
• In addition to supporting the growth of Brucella, the medium is also formulated to promote the luxuriant growth of other fastidious bacteria such as Streptococci, pneumococci, Listeria, Neisseria meningitides, and Haemophilus influenzae. This makes Brucella Agar a useful tool in the laboratory for the isolation and study of these bacteria as well.
• The composition of Brucella Agar includes various ingredients that provide essential nutrients for bacterial growth. Tryptone and peptone serve as sources of nitrogen and carbon, supplying long chain amino acids, vitamins, and other necessary nutrients. Yeast extract acts as a source of vitamin B complex and contributes nitrogenous nutrients. Sodium bisulfite functions as a reducing agent, while sodium chloride helps maintain the osmotic equilibrium of the medium. Dextrose is included as an energy source for bacterial metabolism.
• To enhance the growth of Brucella species, the medium can be further enriched by adding 5% sterile defibrinated horse blood. This addition provides additional nutrients and supports the growth of these bacteria. Additionally, the base medium can be supplemented with freeze-dried antibiotic mixtures to selectively isolate Brucella species.
• Due to the high infectivity of Brucella and the potential risks associated with handling these bacteria, extreme care should be taken when working with Brucella Agar and the organisms it supports. Proper biosafety measures and handling protocols should be followed to ensure the safety of laboratory personnel.
• In summary, Brucella Agar is a specialized medium used for the enrichment, isolation, and cultivation of Brucella and Campylobacter species. It provides the necessary nutrients and conditions for the growth of these fastidious bacteria and can also support the growth of other bacteria with similar requirements. It is an important tool in the laboratory for studying and diagnosing infections caused by these organisms in animals and humans.

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

The principle of Brucella Agar lies in its ability to provide a suitable environment for the growth of fastidious microorganisms. This is achieved through the specific composition of its ingredients.

The peptones present in Brucella Agar serve as a source of organic nitrogen. Fastidious microorganisms often have complex nutritional requirements and rely on organic nitrogen sources for their growth. By providing peptones, Brucella Agar ensures that these organisms have access to the necessary nitrogen compounds they need to thrive.

Yeast extract is another crucial component of Brucella Agar. It acts as a potent source of B-complex vitamins. These vitamins are essential for various metabolic processes in microorganisms, including enzymatic reactions and the synthesis of important cofactors. By supplying B-complex vitamins, the yeast extract in Brucella Agar ensures that fastidious microorganisms have the necessary vitamins for their growth and metabolic activities.

Dextrose, a type of sugar, serves as an energy source in Brucella Agar. Microorganisms utilize dextrose through the process of glycolysis, where it is broken down to produce energy in the form of ATP. By providing dextrose, Brucella Agar ensures that fastidious microorganisms have an easily accessible energy source to fuel their growth and metabolic processes.

Sodium bisulfite is included in Brucella Agar as a reducing agent. It helps to maintain a reduced environment, which is beneficial for the growth of certain microorganisms. A reduced environment provides optimal conditions for the functioning of enzymes and other metabolic processes in these organisms.

Sodium chloride, commonly known as salt, is added to Brucella Agar to maintain the osmotic equilibrium of the medium. Osmotic equilibrium refers to a balanced concentration of solutes inside and outside the cells. By including sodium chloride, Brucella Agar ensures that the osmotic pressure remains at an appropriate level for the growth of fastidious microorganisms.

Finally, agar, a polysaccharide derived from seaweed, serves as the solidifying agent in Brucella Agar. Agar allows the medium to solidify, providing a stable matrix for the microorganisms to grow on. This solidification enables the isolation and examination of individual microbial colonies, making it easier to study and identify specific organisms.

In summary, the principle of Brucella Agar is based on the formulation of its ingredients. Peptones provide organic nitrogen, yeast extract supplies B-complex vitamins, dextrose serves as an energy source, sodium bisulfite acts as a reducing agent, and sodium chloride helps maintain osmotic equilibrium. Agar solidifies the medium, enabling the growth and isolation of fastidious microorganisms for further study and identification.

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 of Use of Brucella Agar

To prepare Brucella Agar, follow the steps below:

1. Suspend 43 grams of the Brucella Agar medium in one liter of purified water. Ensure thorough mixing to achieve a homogenous suspension.
2. Heat the suspension while agitating it frequently. Continue heating until the medium reaches a boiling point and boil for one minute. This step is crucial to completely dissolve the medium and ensure its uniform distribution.
3. After boiling, autoclave the medium at 121°C for 15 minutes. Autoclaving is necessary to sterilize the medium and eliminate any potential contaminants.
4. Once the autoclaving process is complete, the medium is ready for use. Transfer the sterilized Brucella Agar into sterile Petri plates, taking care to maintain aseptic conditions throughout the process.
5. To use the prepared Brucella Agar, standard procedures for obtaining isolated colonies from specimens should be followed. This typically involves inoculating the medium with a small sample or swab from the specimen of interest.
6. Since many pathogens, including Brucella species, require carbon dioxide for their growth during primary isolation, it is important to create an appropriate incubation environment. Incubate the Petri plates at a temperature of 35 ± 2°C for a period of 24 to 72 hours. The incubation should take place in an aerobic atmosphere supplemented with carbon dioxide. This can be achieved by using specialized incubators or gas generators that can provide the required carbon dioxide concentration.

By following these preparation and usage instructions, Brucella Agar can provide a suitable medium for the growth and isolation of fastidious microorganisms, such as Brucella species, from clinical and nonclinical specimens. The incubation conditions, including temperature and carbon dioxide supplementation, create an environment conducive to the growth of these organisms, allowing for their isolation and further study.

Result Interpretation on Brucella Agar

After the incubation period, the interpretation of the results on Brucella Agar involves assessing the growth and characteristics of the colonies present on the plate. Here is a summary of the expected growth and appearance for specific organisms:

1. Brucella melitensis: The colonies of Brucella melitensis will exhibit luxuriant growth. They will appear transparent, raised, convex, with an entire edge, and have a smooth, shiny surface.
2. Brucella ovis: The colonies of Brucella ovis will show good growth and appear convex.
3. Streptococcus pyogenes: Streptococcus pyogenes will exhibit good growth on Brucella Agar.
4. Brucella suis: The colonies of Brucella suis will demonstrate luxuriant growth similar to Brucella melitensis. They will appear transparent, raised, convex, with an entire edge, and have a smooth, shiny surface.
5. Staphylococcus aureus subsp. aureus: The growth of Staphylococcus aureus subsp. aureus may be inhibited on Brucella Agar, meaning that it may not grow or show very limited growth.
6. Escherichia coli: The growth of Escherichia coli may also be inhibited on Brucella Agar, indicating limited or no growth.

When interpreting the results, it is important to observe the characteristics of the colonies, such as their appearance, texture, and edge morphology. This can provide valuable information for the identification and differentiation of different organisms. The growth of each organism can also be semi-quantitatively scored based on the level of growth observed in each streaked area.

In summary, the result interpretation on Brucella Agar involves assessing the growth and characteristics of colonies for different organisms. The expected growth patterns and appearances vary depending on the specific organism, with Brucella species typically exhibiting luxuriant growth, while other organisms may show good growth or be inhibited. Evaluating these colony characteristics aids in the identification and analysis of microorganisms present in the specimen.

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

Brucella Agar has several important uses in the laboratory setting. Here are the key applications of Brucella Agar:

1. Cultivation of Brucella species and fastidious microorganisms: Brucella Agar is primarily used for the cultivation of Brucella species, which are highly fastidious microorganisms. The medium provides the necessary nutrients and conditions for the growth of these bacteria. It is also suitable for the cultivation of other fastidious microorganisms with similar nutritional requirements.
2. General purpose medium for specific bacteria: Brucella Agar serves as a general purpose medium for the cultivation of specific bacteria, including Streptococcus pneumoniae, Streptococcus viridans, and Neisseria meningitidis. These bacteria have specific growth requirements that are met by the composition of Brucella Agar, making it a suitable medium for their cultivation.
3. Determining bacterial hemolytic reactions: Brucella Agar, when supplemented with blood, can be used to determine bacterial hemolytic reactions. By observing the hemolytic patterns produced by bacteria when grown on this medium, such as alpha, beta, or gamma hemolysis, important information about the bacteria’s pathogenicity and virulence can be obtained.
4. Base medium for the isolation of Campylobacter: Brucella Agar can serve as a base medium for the isolation of Campylobacter, a group of bacteria that includes several important pathogens. By adding specific supplements to the Brucella Agar base, it can support the growth and isolation of Campylobacter species from clinical and nonclinical specimens.
5. Isolation of Brucella species from foods: Brucella Agar is recommended by the American Public Health Association (APHA) for the isolation of Brucella species from food samples. This highlights its importance in food safety and surveillance, allowing for the detection and identification of Brucella contamination in food products.
6. Qualitative procedures for the isolation and cultivation of microorganisms: When supplemented with 5% horse blood, Brucella Agar is used in qualitative procedures for the isolation and cultivation of both non-fastidious and fastidious microorganisms. It can be utilized for various clinical and nonclinical specimens, enabling the growth and examination of a wide range of microorganisms.

In summary, Brucella Agar has diverse uses in the laboratory. It serves as a specialized medium for the cultivation of Brucella species and other fastidious microorganisms. Additionally, it functions as a general purpose medium for specific bacteria, aids in determining bacterial hemolytic reactions, and is recommended for the isolation of Campylobacter and Brucella species from various sources, including foods.

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

Quality control of Brucella Agar involves several parameters to ensure its performance and reliability. Here are the key aspects of quality control for Brucella Agar:

1. Appearance: Brucella Agar should appear as a cream to yellow homogeneous free-flowing powder. Any abnormal coloration or inconsistency in the appearance may indicate a quality issue.
2. Gelling: When prepared, Brucella Agar should form a firm gel comparable to a 1.5% Agar gel. The ability of the medium to solidify properly is important for the growth and isolation of microorganisms.
3. Color and clarity of prepared medium: The prepared Brucella Agar should exhibit a yellow color and appear clear to slightly opalescent in Petri plates. Any deviation in color or clarity may suggest a quality problem.
4. pH: The pH of a 4.31% w/v aqueous solution of Brucella Agar should be within the range of 6.80-7.20. The pH level affects the growth and metabolism of microorganisms, so maintaining the specified pH range is crucial for the performance of the medium.
5. Cultural response: After incubation at 35-37°C for 24-72 hours in the presence of 10% CO2, the cultural response of Brucella Agar should be assessed. This includes observing the growth and characteristics of specific organisms used as control strains.
6. Organism growth: The cultural response should be evaluated for control organisms such as Brucella melitensis ATCC 4309 and Brucella suis ATCC 4314. These strains should exhibit luxuriant growth on the Brucella Agar, indicating that the medium supports their growth as expected.
7. Inhibition of certain organisms: The growth of specific organisms, such as Staphylococcus aureus subsp. aureus ATCC 25923 and Escherichia coli ATCC 25922, should be inhibited on Brucella Agar. This serves as a quality control measure, ensuring that the medium is selective for the desired microorganisms.

By carefully monitoring and assessing these quality control parameters, laboratories can ensure that Brucella Agar meets the necessary standards for reliable and consistent results in microbial cultivation and identification.

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

While Brucella Agar is a valuable medium in the laboratory, it does have certain limitations that should be considered. These limitations include:

1. Not intended for diagnostic purposes in humans: Brucella Agar is primarily designed for the cultivation and isolation of microorganisms, particularly Brucella species. It is not intended for direct use in diagnosing diseases or other conditions in humans. Diagnostic tests specific to human clinical specimens should be used for accurate diagnosis.
2. Nutritional variation and growth limitations: Although Brucella Agar provides a nutrient-rich environment suitable for the growth of fastidious microorganisms, there may be variations in nutritional requirements among different strains. Some strains may grow poorly or even fail to grow on this medium. In such cases, alternative culture media and techniques may be necessary to support the growth of these specific strains.
3. Confirmation of anaerobic organisms: Brucella Agar is not specifically formulated for the isolation and identification of anaerobic organisms. If presumptive anaerobic organisms are encountered during culture on Brucella Agar, they should be subjected to confirmatory tests or transferred to appropriate anaerobic culture media to accurately identify them.

It is important to keep these limitations in mind when using Brucella Agar in the laboratory. While it is a useful medium for certain applications, it may not fulfill all the requirements for diagnosing human diseases, and variations in growth among different strains should be considered. Confirmatory tests may be necessary for the identification of certain organisms, especially anaerobes.

FAQ

References

• https://www.mycrobe.org/blog/2018/6/28/brucella-blood-agar
• https://exodocientifica.com.br/_technical-data/M074.pdf
• https://microbiologie-clinique.com/brucella-agar.html
• 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/