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

What is Brucella Agar?

Brucella Agar is a specific type of culture medium that is used to grow Brucella species, which are bacteria that cause brucellosis in people and animals.

It helps hard-to-grow organisms grow by giving them nutrients including peptones, yeast extract, and dextrose.

Made to separate and grow Brucella and other bacteria that don’t need oxygen or only need a little bit of it

Contains lowering chemicals that assist keep oxygen levels low, which is necessary for Brucella to develop.

Used mostly in clinical and veterinary microbiology to find brucellosis by growing blood or tissue samples in a lab

The breakthrough made it easier to find Brucella, which helped with disease management and study into this zoonotic pathogen.

Principle of Brucella Agar

Brucella Agar is a specific type of media that helps Brucella species and other picky bacteria grow.

Casein and meat peptones give bacteria the nitrogen, amino acids, and peptides they need to flourish.

Dextrose is an easy-to-get source of energy.

Yeast extract gives B-complex vitamins and helps things thrive in general.

Sodium chloride keeps the osmotic balance in the medium.

Sodium bisulfite works as a reducing agent, which helps create anaerobic conditions.

The agar is what makes the medium firm and gives it its consistency.

Adding sheep blood gives some anaerobic organisms the growth factors they need and lets aerobic and anaerobic bacteria show haemolysis.

Haemin and Vitamin K1 are added to help picky anaerobes like Prevotella melaninogenica recover and make pigments.

To keep oxidised products from forming before usage, the medium is made in an oxygen-free environment.

Selective variations may have antibiotics such colistin, vancomycin, nitrofurantoin, nystatin, and amphotericin in them to stop other microbes from growing. This helps Brucella species proliferate.

For the best development conditions, the medium is usually kept at 35°C in a microaerophilic atmosphere.

Composition of Brucella Agar

Final pH: 7.0 ± 0.2 at 25°C

Preparation of Brucella Agar

1. Dissolve 43 g of Brucella Agar powder in 1 L of clean water.
2. Stir the mixture often while it heats up and boil it for one minute to fully dissolve.
3. Autoclave at 121°C for 15 minutes to kill germs.
4. If you want to add blood, chill the medium to 45–50°C and add 5% defibrinated sheep or horse blood without getting any germs in it.
5. Put into sterile Petri dishes and let them solidify.
6. Keep prepared plates in a dark place at 2–8°C.

Method of Use of Brucella Agar

• Immunisation
  • Using a sterile loop, directly streak the material onto the surface of Brucella agar.
  • If you’re using a swab, roll it over a tiny area of the agar surface and streak it to separate it.
  • Inoculate selective If there are a lot of other bacteria in the sample, use brucella agar.
• Incubation
  • Put the plates with the inoculation in an incubator at 35°C.
  • Brucella species need CO₂ to grow, therefore put them in a 10% CO₂ atmosphere for 4 to 5 days.
  • If there is no growth, change the CO₂ atmosphere and let it sit for up to 21 days.
• Subculture
  • Check the plates for colonies after they have been incubated.
  • To find out more about questionable colonies, subculture them onto media that don’t select for them.
• Finding
  • Use Gramme staining to see tiny, gram-negative coccobacilli
  • Use biochemical assays like oxidase, catalase, and urea reactions to make sure you have the right Brucella species.
  • Use serological methods to make a definite identification.
• Safety Steps
  • Because Brucella species are infectious, you should only handle cultures in Biosafety Level 3 (BSL-3) circumstances.
  • When working with Brucella cultures, always use biosafety enclosures.
  • To stop infections from spreading in the lab, make careful to throw away all things that have been contaminated with Brucella spp.

Result Interpretation on Brucella Agar

• Brucella species typically form small, round, translucent, non-pigmented colonies on Brucella agar after 48–72 hours of incubation under 5–10% CO₂ at 35–37°C.
• Colony morphology
  • B. melitensis: Small, smooth, convex, glistening, and translucent colonies with entire edges.
  • B. abortus: Similar to B. melitensis, colonies are smooth, convex, and non-hemolytic.
  • B. canis: May exhibit rough or mucoid colonies, which can be sticky and vary in color from white to yellowish or brown.
• Biochemical characteristics
  • Oxidase-positive
  • Catalase-positive
  • Urease-positive
  • Non-hemolytic
  • Non-pigmented
• Identification confirmation
  • Gram staining reveals small, gram-negative coccobacilli.
  • Further identification may require molecular techniques such as PCR or serological tests.

Uses of Brucella Agar

• Isolation and cultivation of Brucella species from clinical and environmental samples
• Support growth of fastidious Brucella bacteria due to enriched nutrients
• Used in diagnosis of brucellosis by culturing blood, bone marrow, or tissue specimens
• Supports growth of other anaerobic and microaerophilic bacteria in clinical microbiology
• Useful for antimicrobial susceptibility testing of Brucella species
• Employed in research studies involving Brucella growth and pathogenicity
• Helps differentiate Brucella colonies by supporting their characteristic morphology and color
• Used in veterinary microbiology for detecting Brucella in animal samples

Precautions

• Use a Class II Biological Safety Cabinet (BSC) – Essential for all manipulations involving Brucella agar to prevent aerosol exposure.
• Wear appropriate personal protective equipment (PPE) – Includes gloves, lab coats, safety goggles, and face shields to minimize direct contact and splashes.
• Avoid procedures that generate aerosols or splashes – Such as pipetting, centrifuging, grinding, blending, shaking, vortexing, mixing, and opening containers of infectious materials.
• Ensure proper ventilation – Maintain good airflow in the workspace to reduce the risk of inhaling airborne particles.
• Do not eat, drink, or smoke in the laboratory – Prevent ingestion or inhalation of potentially infectious material.
• Wash hands thoroughly after handling cultures – Minimizes the risk of contamination and infection.
• Secure and tape shut all culture plates – Reduces the risk of accidental exposure and contamination.
• Store Brucella agar in a cool, dry place – Maintain at temperatures between 2–30°C and protect from sunlight.
• Handle spills and accidents promptly – Follow established protocols for containment and decontamination to prevent exposure.
• Consult with public health authorities if exposure occurs – Seek guidance on post-exposure prophylaxis and monitoring.
• Use Biosafety Level 3 (BSL-3) practices when necessary – Implement BSL-3 precautions when handling products of conception or clinical specimens suspected to contain Brucella.
• Ensure proper disposal of contaminated materials – Autoclave or disinfect before disposal to prevent environmental contamination.
• Maintain an eyewash station and safety shower – Provide immediate access to emergency decontamination facilities.
• Implement a comprehensive biosafety manual – Include protocols for handling, exposure, and emergency procedures.
• Regularly train laboratory personnel – Ensure all staff are aware of and adhere to safety protocols.
• Monitor for symptoms of brucellosis – Conduct regular health checks and serological testing for exposed individuals.
• Limit access to the laboratory – Restrict entry to authorized personnel to minimize exposure risk.
• Use mechanical pipetting devices – Avoid mouth pipetting to reduce the risk of accidental ingestion.
• Decontaminate work surfaces regularly – Use effective disinfectants to clean surfaces after handling Brucella agar.
• Ensure proper labeling and storage of cultures – Clearly mark all containers and store them securely to prevent accidental exposure.
• Avoid direct contact with contaminated materials – Use tools and equipment to handle potentially infectious substances.
• Report any exposure incidents immediately – Follow institutional protocols for reporting and managing exposures.
• Regularly review and update safety protocols – Ensure that all procedures are current and effective in minimizing risk.
• Consult safety data sheets (SDS) for specific products – Obtain detailed information on handling and hazards associated with Brucella agar.
• Implement a spill response plan – Ensure all personnel are trained in procedures for managing spills involving Brucella agar.
• Maintain records of training and exposure incidents – Document all safety training and any exposure events for compliance and review.
• Ensure proper waste disposal procedures – Follow local regulations for the disposal of biological waste to prevent environmental contamination.
• Use appropriate containers for waste – Place contaminated materials in leak-proof, puncture-resistant containers for disposal.
• Perform regular audits of safety practices – Conduct inspections to ensure compliance with biosafety protocols.
• Maintain an emergency contact list – Ensure that all personnel have access to contact information for emergency responders and public health authorities.
• Ensure proper labeling of all cultures and reagents – Clearly mark all materials with relevant information to prevent confusion and ensure safe handling.
• Limit the use of sharps – Use alternatives to needles and other sharp instruments when possible to reduce the risk of injury.
• Use mechanical devices for transferring cultures – Employ tools such as inoculating loops or automatic pipettes to handle cultures safely.
• Maintain a clean and organized laboratory environment – Ensure that work areas are free from clutter to reduce the risk of accidents.
• Implement a labeling system for reagents and cultures – Use color-coded or clearly marked labels to identify materials and their hazards.
• Ensure proper ventilation in all laboratory areas – Maintain airflow systems to prevent the accumulation of hazardous fumes or aerosols.
• Use appropriate disinfectants for decontamination – Select disinfectants effective against Brucella species for cleaning surfaces and equipment.
• Ensure that emergency equipment is accessible and functional – Regularly check eyewash stations, safety showers, and first aid kits to ensure they are ready for use.
• Provide adequate lighting in all laboratory areas – Ensure that workspaces are well-lit to prevent accidents and facilitate accurate work.
• Implement a system for reporting safety concerns – Encourage personnel to report potential hazards or unsafe practices to management.
• Conduct regular safety drills – Practice emergency procedures to ensure that all personnel are prepared for potential incidents.
• Maintain an inventory of all hazardous materials – Keep records of all chemicals and biological agents used in the laboratory for safety and regulatory compliance.
• Ensure compliance with local and international regulations – Stay informed about and adhere to laws and guidelines governing laboratory safety and biosafety.
• Provide ongoing education and training – Offer regular updates and refresher courses on laboratory safety practices and procedures.
• Implement a system for tracking and managing laboratory equipment – Ensure that all equipment is properly maintained and calibrated to prevent malfunctions.
• Maintain a clean and organized laboratory environment – Regularly clean and disinfect work areas to prevent contamination and accidents.
• Ensure proper disposal of laboratory waste – Follow established protocols for the disposal of biological and chemical waste to prevent environmental contamination.

Storage condition of Brucella Agar

• Storage Temperature
  • 2–30°C
  • 15–25°C
  • 2–8°C
  • Below 30°C
  • Below 8°C
• Storage Conditions
  • Keep in a cool, dry place
  • Protect from direct sunlight and heat
  • Store in original, tightly sealed containers
  • Avoid freezing or overheating
  • Store in a well-ventilated area
  • Keep away from incompatible substances
  • Do not use if there are signs of deterioration (e.g., discoloration, cracking)
• Shelf Life
  • 90 days from date of manufacture
  • 3 months
  • Use before the expiration date printed on the product label

Quality Control of Brucella Agar

Following organisms can be commonly utilized for quality control testing in Anaerobe Systems.

Limitations of Brucella Agar

• Slow growth of Brucella spp. may require prolonged incubation up to 21–42 days, reducing diagnostic speed and sensitivity.
• Non-selectivity allows contaminants: fungal and Gram-positive bacteria frequently grow on Brucella agar, potentially overgrowing Brucella colonies.
• Fungal contamination particularly high due to absence of antifungal agents like cycloheximide or amphotericin B.
• Not fully selective—other organisms (e.g., Proteus spp., Clostridium spp.) can swarm and mask target growth.
• Incomplete inhibition of enteric bacilli in dense inocula; selective variants or supplementary media often needed.
• Poorer performance in AST for certain anaerobes (e.g., C. difficile) compared to optimized media; may yield suboptimal growth or MIC values
• Requires confirmatory tests and additional media for full identification—cannot serve as a stand-alone diagnostic medium.
• Safety risk: culture involves biosafety level 3 practices; aerosolization from plates poses hazard to lab personnel

FAQ