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

What is Brucella Agar?

Brucella agar’s a nutritionally rich solid medium used mainly for the isolation of Brucella species, tho it also supports fastidious organisms like Bacteroides and Fusobacterium. it contains peptones, yeast extract, glucose, sodium chloride—those provide nitrogenous compounds, vitamins, energy, osmotic balance… all critical for bacterial metabolism. the pH is usually adjusted to 7.0 ± 0.2, if it’s off, Brucella won’t grow well. sometimes labs add 5–10% defibrinated sheep blood to enrich the medium, helps with Brucella recovery, esp. from blood or tissue samples.

Selective versions may include basic fuchsin, thionin, or antibiotics like polymyxin B, bacitracin—to suppress contaminants or differentiate Brucella strains based on dye tolerance. colonies are typically small, round, convex, and kinda translucent, sometimes sticky, no pigment mostly. it’s incubated at 35–37°C, under 5–10% CO₂, and growth shows up in 48–72 hrs, though some take longer. used in both clinical and vet labs, and research. but it ain’t fully selective unless modified, so overgrowth from other bacteria can still happen. handling requires BSL‑3 precautions, cause Brucella’s airborne risk is real—lot of lab-acquired infections reported when plates opened carelessly or incubators not sealed proper.

Principle of Brucella Agar

Brucella agar operates as an enriched and semi‑selective medium, designed primarily to promote growth of fastidious organisms, esp. Brucella spp., while inhibiting widespread contaminants.

The nutrient‑rich base contains enzymatic digests of casein and animal tissue (about 10 g/L each), yeast extract (2 g/L), dextrose (1 g/L), sodium chloride (5 g/L), sodium bisulfite (~0.1 g/L) and agar (13–15 g/L), all at pH ~7.0 ± 0.2.

Peptones supply organic nitrogen for protein synthesis, yeast extract yields B‑complex vitamins critical as enzyme cofactors, dextrose fuels metabolism via glycolysis, while sodium chloride maintains osmotic balance.

Sodium bisulfite acts as a reducing agent, lowering oxidation‑reduction potential, which aids survival of microaerophilic organisms like Brucella or anaerobes.

Blood enrichment (typically 5–10 % defibrinated sheep or horse blood) supplies additional growth factors like hemin and NAD (X and V factors), enriching medium for Brucella and enhancing recovery of anaerobic flora.

For selectivity, antibiotics (e.g. bacitracin, polymyxin B, cycloheximide) or dyes like thionin/fuchsin may be added post cooling to suppress competing flora and allow differentiation of Brucella species based on dye sensitivity.

The combined principle: nutrient‑rich, reducing, enriched, and optionally selective, thereby permitting Brucella to grow slowly over 48–72 h in 5–10% CO₂ while minimizing background flora and supporting strict pathogens

Composition of Brucella Agar

Final pH: 7.0 ± 0.2 at 25°C

Preparation of Brucella Agar

1. Measure the base – weigh exactly 41 grams of Brucella Agar powder, not more not less, for every 1 liter distilled water.
2. Dissolve the powder – add the powder slowly into boiling distilled water, stir constantly, if it clumps, the mix won’t be even, that ruins it.
3. pH adjustment – check pH, adjust to 7.0 ± 0.2, use meter or strip, but don’t skip it—wrong pH messes up bacterial growth.
4. Sterilization step – autoclave at 121°C for 15 minutes, pressure 15 psi, if overheated, it’ll break down nutrients, esp. glucose.
5. Cooling stage – after autoclave, cool medium to about 45–50°C, don’t let it drop below 40 or it starts gelling too early.
6. Add blood if needed – for enriched version, aseptically add 5–10% defibrinated sheep blood, mix gently, don’t shake it.
7. Optional additives – if selective version needed, add thionin, fuchsin, or antibiotic mix (like polymyxin-B, bacitracin), only after cooling.
8. Plate pouring – pour ~20 ml per sterile petri dish, use biosafety cabinet or clean area, let solidify without covering immediately.
9. Label & date – every plate needs date, media name, batch no., written clearly on bottom, or things get misidentified later.
10. Storage – keep plates at 2–8°C, sealed tight to avoid drying, best used within 2–3 weeks, after that quality drops.

Method of Use of Brucella Agar

1. sample collection – take clinical sample like blood, bone marrow, tissue, whatever required, but make sure it’s sterile n fresh… delays affect viability.
2. plate labeling – write ID, date, initials on bottom of petri dish, not lid—people mess that up all the time, leads to wrong results.
3. inoculation method – streak sample using sterile loop, use 3-zone method or full streak, just don’t overdo pressure, it’ll gouge the agar.
4. optional pre-enrichment – for weak samples, pre-incubate in Brucella broth or Castaneda media, usually 24 hrs at 37°C, then subculture.
5. incubation setup – place plates inside CO₂ incubator, needs 5–10% CO₂, temp should be steady 35–37°C, not room temp, that won’t work.
6. incubation time – check after 48 hrs, but don’t toss it till 7 days—Brucella grows slow, like real slow, colonies tiny at first.
7. colony observation – look for small, smooth, convex colonies, kinda sticky, color mostly translucent, maybe pale gray-white, not flashy.
8. blood agar sign – usually no hemolysis, but few strains might give faint alpha… hard to see unless you know what to look.
9. confirmation step – pick colony, gram stain it, should show gram-neg coccobacilli, short chains or clumps, then do oxidase, urease, maybe PCR if lab got it.
10. waste disposal – used plates, loops, gloves, everything—autoclave at 121°C for 30 mins, then dump in biohazard bin, no shortcuts here.
11. log data – record morphology, growth time, any hemolysis, confirm tests—don’t rely on memory, gotta write down everything.

Result Interpretation on Brucella Agar

Brucella spp. growth – shows small, round, convex colonies, usually translucent or pale yellow, after 48–72 hrs incubation.

Hemolysis observation – on blood-supplemented Brucella agar, some strains may show alpha or beta hemolysis, though many don’t.

Selective media response – when antibiotics or dyes (like basic fuchsin or thionin) are present:

• Brucella abortus – usually inhibited by basic fuchsin, but resistant to thionin.
• Brucella melitensis – resistant to both fuchsin and thionin.
• Brucella suis – usually inhibited by thionin, but may tolerate fuchsin.

Contaminants – fast-growing organisms (like E. coli, Staph, or fungi) form large, opaque, pigmented colonies, not typical.

No growth – may indicate:

• medium expired or poorly storedincorrect incubation (temp, CO₂ level wrong)
• non-viable organism sample or wrong ID

Microscopic view – gram stain from colonies should show gram-negative coccobacilli, often in pairs or short chains, faint staining.

Colony texture – slightly sticky or smooth, may differ by species or growth phase.

Interpretation caution – always confirm Brucella spp using biochemical tests, serological ID, or PCR, ’cause false positives can happen.

Colony morphology on Brucella Agar

• Colony size – usually small, about 0.5–1.5 mm, takes 48–72 hrs to fully appear, slower than most bacteria.
• Shape & edges – colonies show round, convex shape, smooth margin, not fuzzy or spreading.
• Elevation – typically raised or low convex, not flat, sometimes hard to spot if lighting poor.
• Surface texture – smooth, sometimes moist-looking or a bit glossy, not rough or wrinkled.
• Color – colonies look translucent to grayish-white, rarely pale yellow, depends on blood addition and strain.
• Consistency – colonies often feel buttery or slightly sticky, not dry when touched with sterile loop.
• Hemolysis (on blood agar) – mostly non-hemolytic, though few strains may show weak alpha hemolysis, often faint or missed.
• Odor – almost none, maybe faint earthy smell, but never sharp or sweet like Pseudomonas or Proteus.
• Gram stain from colony – shows Gram-negative coccobacilli, arranged in pairs or small clumps, stains faint, sometimes easy to under-read.

Uses of Brucella Agar

• Isolation of Brucella spp. – used mainly for recovering Brucella from blood, bone marrow, lymph, or other infected tissue.
• Anaerobe cultivation – supports fastidious anaerobes, like Bacteroides, Fusobacterium, even some Clostridium spp.
• Veterinary diagnostics – applied in detecting Brucella infection in livestock, esp. cattle, goats, pigs, where zoonotic concern exists.
• Selective identification – when supplemented with thionin or fuchsin, helps differentiate Brucella species based on dye resistance.
• Campylobacter recovery – blood-enriched version supports Campylobacter jejuni & other microaerophilic pathogens.
• Clinical research – used in labs studying intracellular pathogens, immune evasion, or slow-growing gram-negatives.
• Biochemical profiling base – colonies grown on Brucella agar often used for downstream oxidase, urease, catalase or molecular testing.
• Quality control testing – in media manufacturing, used to check growth-support ability for fastidious organisms.
• Antibiotic resistance testing – sometimes applied for in-vitro susceptibility profiling of Brucella, esp. in outbreak studies.

Precautions

• BSL-3 handling – brucella considered super infectious, so work needs biosafety level 3 facility, if not, don’t even start, serious risk.
• Aerosol risk – avoid vortexing or pipetting fast, ’cause even a tiny splash or mist spreads Brucella, many lab workers got infected like this.
• No open flames – don’t use flame near plates, it causes convection currents and spreads aerosols instead of sterilizing.
• Disinfect spills fast – if anything spills, pour 1% hypochlorite or phenol, wait 20–30 min, don’t rush to wipe or you’ll smear it more.
• Full PPE always – wear gloves, long-sleeve gown, face mask or respirator, goggles too, not just lab coat and think it’s fine.
• Seal and sterilize waste – all used plates or pipette tips must be autoclaved, minimum 121°C for 30 mins, else Brucella survives.
• Check plates before use – if condensation on lid or weird smell or visible fungal dots—throw it, even tiny cracks ruin sterility.
• Avoid cross-contam – use separate tools for different samples, if loop touches multiple plates, you already messed up the results.
• Incubation safety – don’t open incubator while plates still venting gas, Brucella may escape in CO₂ puff—wait or open slow.
• Label biohazard clearly – every petri dish, tube, or bag with Brucella must show bright red BIOHAZARD, else it gets mishandled by staff.

Storage condition of Brucella Agar

• Unprepared powder – store Brucella agar base powder in tight, moisture-proof container, temp should stay 15–30°C, away from direct light or humidity.
• Prepared medium (without blood) – after autoclaving, store in sterile bottles at 2–8°C, keep for max 4 weeks, if not contaminated.
• Prepared plates (with blood) – store petri plates at 2–8°C, away from light, use within 1–2 weeks, since blood deteriorates fast.
• Avoid freezing – never freeze agar plates or slants, it messes up texture, cracks the gel, causes weird growth patterns.
• Label all batches – every batch must have prep date, expiry, batch no., else forget which one’s still valid.
• Protect from drying – store plates in sealed plastic sleeves or containers, if they dry out, Brucella won’t grow proper.
• Don’t stack too high – while storing, stack plates 5–10 max, or condensation forms, messes up surface, colonies slide.
• Check before use – inspect for cracks, moisture pools, color changes, any of that—discard, even if expiry not reached.

Quality Control of Brucella Agar

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

Limitations of Brucella Agar

• Non-selective base – plain Brucella agar doesn’t stop contaminants or normal flora, so false colonies may outgrow Brucella if no antibiotics added.
• Slow growth – Brucella spp grow slow, like 48–72 hrs or more, so fast growers (like E. coli or molds) may mask colonies.
• Needs CO₂ – not useful in labs without CO₂ incubators, ’cause Brucella needs 5–10% CO₂ or else growth gets patchy or fails.
• No species ID – can’t differentiate Brucella species unless dyes like thionin or fuchsin added, otherwise all look same.
• Blood-dependent – enriched growth needs defibrinated blood, which adds cost, handling risk, also shortens shelf-life.
• No definitive diagnosis – colonies need confirmation via biochem, serology or PCR, alone it can’t confirm Brucella, many bacteria show similar growth.
• Prone to desiccation – media dries easily if not stored airtight, once dry, agar surface cracks, growth gets irregular.
• Biohazard risk – Brucella agar work poses high infection risk, so can’t be safely used in low-resource or non-BSL3 labs.

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