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Drugs for Tuberculosis - Complete MBBS Pharmacology

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Overview: Why Multiple Drugs?

1. Actively dividing extracellular bacilli (in lung cavities) - killed by isoniazid, rifampin
2. Slowly dividing intracellular bacilli (inside macrophages) - killed by pyrazinamide
3. Dormant/persister bacilli (in acidic granuloma environment) - killed by pyrazinamide
4. Semi-dormant bacilli (caseous foci) - killed by rifampin

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Classification of Anti-TB Drugs

FIRST-LINE DRUGS (RIPE mnemonic)

SECOND-LINE DRUGS

• Injectable aminoglycosides: Amikacin, Capreomycin, Kanamycin
• Fluoroquinolones: Moxifloxacin, Levofloxacin
• Oral bacteriostatic agents: Ethionamide, Cycloserine, PAS (para-aminosalicylic acid)
• Novel agents: Bedaquiline, Linezolid, Delamanid, Pretomanid, Clofazimine

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FIRST-LINE DRUGS (Detailed)

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1. ISONIAZID (INH / H)

• INH is a prodrug - activated by mycobacterial KatG catalase-peroxidase
• The activated INH-NADH complex inhibits InhA (mycobacterial ketoenoyl-reductase / enoyl-ACP reductase)
• This blocks mycolic acid synthesis - mycolic acids are essential long-chain fatty acids in the mycobacterial cell wall
• KatG activation also releases free radicals (including nitric oxide) that have direct antimycobacterial activity
• Selective toxicity: Human cells lack mycolic acid synthesis, so INH has no toxicity to human cells via this mechanism

• Excellent oral absorption (peak serum: 3-5 mcg/mL in 30 min-2 h)
• Widely distributed including CSF (CSF levels = serum levels) - important for TB meningitis
• Metabolized in liver by N-acetyltransferase 2 (NAT2) - acetylation + hydrolysis
• Polymorphic acetylation (genetically determined):
  • Slow acetylators (most Indians, Europeans) - higher drug levels, more toxicity (neuropathy)
  • Fast acetylators (East Asians) - lower drug levels, may need higher doses
• Inhibits CYP450 - increases levels of warfarin, carbamazepine, phenytoin

• Adults: 5 mg/kg/day (max 300 mg/day)
• Children: 10-15 mg/kg/day
• Intermittent: 15 mg/kg twice weekly (max 900 mg)
• Always give with pyridoxine 25-50 mg/day to prevent neuropathy

• 5 mechanisms identified:
  • katG mutations (most common - impairs prodrug activation)
  • inhA mutations (target modification - also causes low-level resistance to ethionamide)
  • kasA, NADH dehydrogenase 2 mutations
  • Efflux pumps (efpA, mmpL7, Rv1258c) in 20-30%
• ~7-10% of US isolates resistant

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2. RIFAMPIN (Rifampicin / R / RIF)

• Semisynthetic derivative of rifamycin (from Amycolatopsis rifamycinica)
• Binds β-subunit of bacterial DNA-dependent RNA polymerase → inhibits RNA transcription
• Bactericidal - active against both intracellular and extracellular organisms
• Penetrates phagocytic cells, abscesses, and lung cavities effectively
• Human RNA polymerase is not inhibited (selectivity)

• Well absorbed orally (take on empty stomach - food reduces absorption)
• Excreted mainly via bile → enterohepatic recirculation → feces (deacetylated metabolite)
• Small amount excreted in urine
• No dose adjustment in renal impairment
• Imparts orange-red color to urine, sweat, tears, saliva (warn patient - harmless)

• 600 mg/day (or 10 mg/kg/day) orally
• For LTBI (latent TB): 600 mg/day x 4 months as monotherapy (preferred over INH for LTBI)

• Oral contraceptives (OCP failure - use barrier method)
• Warfarin (reduced anticoagulation)
• Antiretrovirals (protease inhibitors, NNRTIs, integrase inhibitors)
• Cyclosporine, tacrolimus (organ rejection risk)
• Methadone (precipitates withdrawal)
• Anticonvulsants (phenytoin levels fall)
• Oral hypoglycemics

• Point mutations in rpoB gene (β-subunit of RNA polymerase)
• Complete cross-resistance within all rifamycins (rifampin, rifabutin, rifapentine)
• Rifampin resistance = marker for MDR-TB (since INH+RIF combination is the backbone)

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3. PYRAZINAMIDE (PZA / Z)

• Nicotinamide analogue - prodrug
• Converted by mycobacterial pyrazinamidase (encoded by pncA gene) to active pyrazinoic acid (POA)
• POA disrupts mycobacterial cell membrane metabolism and transport; fatty acid synthase I is likely the primary target
• Active only in acidic environment (pH <6.0) - the acidic milieu inside macrophage lysosomes and within caseous granulomas
• Therefore kills the intracellular "persister" population - no other drug does this as effectively
• This is why adding PZA to the initial 2-month phase allows treatment to be shortened from 9 months to 6 months

• Well absorbed orally; peak serum: 20-60 mcg/mL at 1-2 h
• Widely distributed including CSF (important for TB meningitis)
• Metabolized in liver; metabolites cleared renally
• Dose reduction needed in renal impairment (CrCl <30 mL/min - give 3x weekly, not daily)

• 25 mg/kg/day (max 2 g/day) or 15-30 mg/kg/day

• Mutations in pncA gene (72-98% of resistant strains) → impaired pyrazinamidase → can't convert PZA to active POA

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4. ETHAMBUTOL (EMB / E)

• Bacteriostatic (only first-line drug that is bacteriostatic - the rest are bactericidal)
• Inhibits arabinosyltransferases (embB gene) involved in mycobacterial cell wall synthesis
• Specifically inhibits formation of arabinogalactan and lipoarabinomannan (components of the mycobacterial cell wall)
• MIC: 0.5-2 mcg/mL

• 75-80% absorbed orally; peak serum 2-4 mcg/mL at 2-4 h
• Well distributed but poorly penetrates CSF (needs 25 mg/kg for CSF levels)
• Mainly excreted unchanged in urine - dose reduction in renal impairment

• Intensive phase: 15-25 mg/kg/day
• The 4th drug in the RIPE regimen - primarily included to prevent resistance if INH or RIF resistance is present

• Missense mutations in embB gene (codon 306 in 50-70%)

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STANDARD TREATMENT REGIMENS

Drug-Susceptible Pulmonary TB (6-month standard regimen)

INTENSIVE PHASE (2 months): HRZE daily
    ↓
CONTINUATION PHASE (4 months): HR daily

• HRZE = Isoniazid + Rifampin + Pyrazinamide + Ethambutol
• Ethambutol can be dropped if susceptibility to INH + RIF is confirmed
• INH-RIF alone for 9 months cures 95-98% of susceptible TB

Newer 4-Month Regimen (2022 onward)

INTENSIVE PHASE (8 weeks): RPT + MOX + INH + PZA (daily)
    ↓
CONTINUATION PHASE (9 weeks): RPT + MOX + INH (daily)

• Rifapentine + Moxifloxacin + Isoniazid + Pyrazinamide
• Non-inferior to 6-month regimen for adults/adolescents ≥40 kg

Latent TB Infection (LTBI)

• Rifampin alone x 4 months - preferred, most effective
• INH alone x 6-9 months - alternative
• INH + Rifapentine weekly x 12 weeks (3HP regimen) - for contacts

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SECOND-LINE DRUGS (Detailed)

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FLUOROQUINOLONES (Moxifloxacin, Levofloxacin)

• Mechanism: Inhibit DNA gyrase (topoisomerase II) and topoisomerase IV → block DNA replication
• Moxifloxacin is the most potent anti-TB fluoroquinolone (now first-line in 4-month regimen)
• Used in MDR-TB and drug-intolerant patients
• ADR: QT prolongation (moxifloxacin), tendinopathy, CNS effects

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BEDAQUILINE

• First new TB drug in 40 years (FDA approved 2012)
• Mechanism: Inhibits mycobacterial ATP synthase (blocks energy production)
• Bactericidal against both replicating AND non-replicating bacilli
• Used in MDR-TB and XDR-TB regimens
• ADR: QT prolongation (most important - ECG monitoring mandatory), hepatotoxicity, nausea

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AMINOGLYCOSIDES (Amikacin, Kanamycin, Capreomycin)

• Mechanism: Bind 30S ribosomal subunit → inhibit protein synthesis (amikacin); capreomycin is a cyclic peptide with similar mechanism
• Injectable agents - used in MDR-TB
• ADR: Nephrotoxicity, ototoxicity (8th nerve damage - vestibular + cochlear)

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ETHIONAMIDE

• Mechanism: Structural analogue of INH; also a prodrug, also inhibits InhA (mycolic acid synthesis) - activated by EtaA monooxygenase (not KatG)
• Cross-resistance with INH (inhA mutations) but not katG mutations
• Dose: 500-750 mg/day in divided doses
• ADR: Severe GI intolerance (nausea, vomiting - dose-limiting), hepatotoxicity, hypothyroidism, metallic taste, peripheral neuropathy

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CYCLOSERINE

• Mechanism: Analogue of D-alanine; inhibits alanine racemase and D-Ala-D-Ala ligase → blocks peptidoglycan cell wall synthesis
• Dose: 500-1000 mg/day in divided doses
• ADR: CNS toxicity - seizures, psychosis (suicide risk), peripheral neuropathy, somnolence
• Monitor drug levels; supplement pyridoxine
• Contraindicated in epilepsy, severe renal insufficiency, active alcohol use, depression history

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PAS (Para-aminosalicylic acid)

• Mechanism: Structural analogue of PABA; inhibits folate synthesis in mycobacteria (similar to sulfonamides)
• Also may inhibit iron uptake by mycobacteria
• Dose: 8-12 g/day in divided doses
• ADR: Severe GI intolerance, hepatotoxicity, hypothyroidism (interferes with iodine incorporation), hypersensitivity, malabsorption syndrome

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LINEZOLID

• Mechanism: Oxazolidinone; inhibits 50S ribosome assembly (unique binding site - prevents initiation complex formation)
• Active against MDR-TB and XDR-TB; excellent intracellular penetration
• Part of BPaL regimen (Bedaquiline + Pretomanid + Linezolid) for XDR-TB
• Dose: 600 mg/day (1200 mg/day for first 6 months in some protocols)
• ADR: Bone marrow suppression (anemia, thrombocytopenia), irreversible peripheral and optic neuropathy (prolonged courses), serotonin syndrome (if combined with serotonergic agents)
• Monitor CBC; supplement pyridoxine

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RIFABUTIN

• Semisynthetic rifamycin; same mechanism as rifampin (inhibits RNA polymerase)
• Complete cross-resistance with rifampin
• Key advantage: Less potent CYP450 inducer than rifampin
• Used instead of rifampin in HIV-TB co-infection - especially when patient is on protease inhibitors or NNRTIs (rifabutin does not drop PI levels as drastically)
• Dose: 300 mg/day (reduce by half with PIs; increase to 600 mg/day with efavirenz)
• ADR: Hepatotoxicity, rash, leukopenia, thrombocytopenia, optic neuritis, uveitis (unique to rifabutin)

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DELAMANID & PRETOMANID (Nitroimidazoles)

• Mechanism: Prodrugs activated by mycobacterial flavin-dependent nitroreductases → inhibit mycolic acid biosynthesis + generate nitric oxide and reactive oxygen species
• Delamanid: Used in MDR-TB (children <6 years with rifampicin-resistant TB); 100 mg twice daily
• Pretomanid: Part of BPaL regimen for XDR-TB; 200 mg/day
• ADR: QT prolongation (delamanid especially), hepatotoxicity (pretomanid in combination)

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CLOFAZIMINE

• Riminophenazine dye; primarily used in leprosy but increasingly in MDR-TB
• Mechanism: Increases reactive oxygen species, membrane destabilization; also anti-inflammatory
• Half-life: ~70 days
• ADR: GI intolerance, reversible orange-brown discoloration of skin and secretions

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MDR-TB and XDR-TB Treatment

• MDR-TB: Resistant to at least INH + Rifampin
• XDR-TB (formerly Pre-XDR + XDR): MDR-TB + resistance to fluoroquinolones ± injectable agents

• BPaZLfx: Bedaquiline + Pretomanid + Pyrazinamide + Linezolid + Levofloxacin
• BPaL: Bedaquiline + Pretomanid + Linezolid (for XDR-TB - Nix-TB trial)
• Duration: 18-20 months

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High-Yield Memory Tricks (MBBS Exam)

• Cell wall synthesis blockers: INH (mycolic acid), Ethambutol (arabinogalactan), Cycloserine (peptidoglycan), Delamanid/Pretomanid (mycolic acid via nitroreductases)
• RNA polymerase inhibitor: Rifampin (and all rifamycins)
• Protein synthesis inhibitor: Aminoglycosides (30S), Linezolid (50S)
• ATP synthase inhibitor: Bedaquiline
• Folate synthesis inhibitor: PAS
• Membrane disruptors: Pyrazinamide (in acidic pH), Clofazimine

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Special Situations

• Use rifabutin instead of rifampin (less drug interaction with ARVs)
• Efavirenz-based ART: rifampin can be used (efavirenz dose may need increase)
• Integrase inhibitors (dolutegravir): use rifabutin (rifampin reduces dolutegravir levels significantly)

• HRZE is acceptable (first-line regimen)
• Streptomycin is contraindicated (ototoxicity to fetus)
• Pyrazinamide: controversial in USA, used by WHO
• Add pyridoxine throughout

• All drugs except ethambutol (poor CSF penetration) penetrate CSF well
• Use INH, RIF, PZA as backbone; add fluoroquinolone or ethionamide if needed
• Duration extended to 9-12 months

• All first-line drugs are hepatotoxic; monitor LFTs
• Stop if ALT >3x ULN with symptoms, or >5x ULN without symptoms
• Streptomycin + ethambutol can replace hepatotoxic drugs temporarily

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MBBS Microbiology University Practicals - Complete Guide

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PRACTICAL SYLLABUS OVERVIEW

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SECTION 1: MICROSCOPY

Types of Microscopes Used in Microbiology

• Q: What objective lens is used for oil immersion? A: 100x (total magnification 1000x)
• Q: Why is immersion oil used? A: Same refractive index as glass - prevents light from scattering, increases resolution
• Q: What is the resolving power of a light microscope? A: ~0.2 micrometers (200 nm)
• Q: Which microscope is used for Treponema? A: Dark field - T. pallidum cannot be cultured or stained by ordinary methods

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SECTION 2: STAINING TECHNIQUES

PRACTICAL 1: GRAM STAIN

Principle

• Gram-positive bacteria: thick peptidoglycan layer (20-80 nm) retains the crystal violet-iodine complex after decolorization
• Gram-negative bacteria: thin peptidoglycan (2-7 nm) + outer lipopolysaccharide membrane; lipid layer dissolves with alcohol, releasing the crystal violet-iodine complex

Reagents Required

1. Crystal violet (primary stain) - basic dye
2. Gram's iodine (mordant - fixes the dye)
3. Acetone-alcohol 30% (decolorizer)
4. Safranin (counterstain, red)

Step-by-Step Procedure

Results

• Gram-positive bacteria: Purple/Violet
• Gram-negative bacteria: Pink/Red

Examples of Results

Common Errors and How to Avoid

Viva Points - Gram Stain

• Q: What is the mordant in Gram stain? A: Gram's iodine - it forms an insoluble crystal violet-iodine complex with the primary stain
• Q: Why do Gram-negative bacteria stain pink? A: Their outer lipid membrane dissolves with alcohol, releasing crystal violet; they then take up the safranin counterstain
• Q: Name a Gram-variable organism. A: Mycobacterium (does not stain well with either), Gardnerella vaginalis
• Q: Why can't Mycobacterium be Gram stained? A: Mycolic acid in cell wall is waxy and impermeable to crystal violet; needs special acid-fast stain
• Q: Which bacteria are inherently Gram-variable? A: Mycoplasma (no cell wall), L-forms

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PRACTICAL 2: ZIEHL-NEELSEN (ZN) ACID-FAST STAIN

Principle

Reagents Required

1. Carbolfuchsin (primary stain) - basic fuchsin + phenol
2. 3% acid-alcohol (3% HCl in 95% ethanol) - decolorizer
3. Löffler's methylene blue (counterstain)

Step-by-Step Procedure (Hot Method - Ziehl-Neelsen)

Results

• Acid-fast bacilli (AFB): Red/Bright pink rods on blue background
• Non-acid-fast organisms: Blue (take counterstain)

Cold Method - Kinyoun Stain

• No heat required - uses higher concentration of carbolfuchsin (4g basic fuchsin + 8g phenol)
• Stain for 3 min (no heating), then proceed as ZN
• Advantage: No risk of boiling or fire; suitable for biosafety cabinets

Modified ZN Stain (1% H2SO4 decolorizer instead of acid-alcohol)

• Uses 1% sulfuric acid as decolorizer (weaker than HCl-alcohol)
• Used for partially acid-fast organisms: Nocardia, Cryptosporidium, Isospora
• Mycobacteria = strong acid-fast; Nocardia = weakly acid-fast

Auramine-Rhodamine (Fluorescent) Stain for AFB

• Auramine + rhodamine bind to mycolic acids; fluoresce under UV
• Most sensitive method for detecting AFB in sputum
• Positive fluorescent result confirmed by ZN stain
• AFB appear bright yellow-orange on dark background

Reporting AFB Smear Results

Viva Points - ZN Stain

• Q: Why is heat used in ZN stain? A: Heat drives carbolfuchsin into the waxy mycolic acid layer of mycobacteria
• Q: Which organisms are acid-fast? A: Mycobacterium tuberculosis, M. leprae, M. avium; Nocardia (weakly); Cryptosporidium, Isospora oocysts (modified ZN)
• Q: What is the decolorizer in ZN stain? A: 3% HCl in 95% ethanol (acid-alcohol)
• Q: Difference between ZN and Kinyoun stain? A: Kinyoun is a cold method (no heat) using higher carbolfuchsin concentration
• Q: Why is ZN stain not 100% sensitive for TB? A: Requires ~5,000-10,000 bacilli/mL of sputum to give a positive result; fluorescent stain is more sensitive

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PRACTICAL 3: OTHER STAINING METHODS

Albert's Stain (for Corynebacterium diphtheria - Metachromatic Granules)

1. Prepare and heat-fix smear
2. Apply Albert's stain for 3-5 minutes
3. Rinse with water
4. Apply Gram's iodine for 1 minute (mordant)
5. Rinse, dry, examine

• Cell body: Green/bluish-green
• Metachromatic granules: Dark blue/black (at poles of bacilli)
• Typical appearance: "Chinese letter" or "cuneiform" arrangement

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Capsule Stain (Anthony's Method / Negative Staining)

1. Mix sample with 1 drop crystal violet on slide; spread, do NOT heat fix (heat destroys capsule)
2. Apply copper sulfate solution
3. Air dry, examine

• Capsule: Unstained clear/colorless halo around purple cell body
• Cell: Dark purple
• Background: Light blue-green (copper sulfate)

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Spore Stain (Schaeffer-Fulton Method)

1. Heat-fix smear
2. Flood with 5% malachite green, heat over steam for 5 min
3. Wash with water for 30 seconds (removes stain from vegetative cells)
4. Counterstain with 0.5% safranin for 30 sec
5. Wash, dry, examine

• Spores: Green (retain malachite green)
• Vegetative cells: Pink/Red (take safranin)

• Central oval spore: B. anthracis, B. cereus
• Subterminal: C. tetani - no; C. perfringens - central
• Terminal round (drumstick): C. tetani
• Subterminal oval (bulging): C. botulinum, C. difficile

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Flagella Stain

• Monotrichous: Single polar flagellum (V. cholerae)
• Lophotrichous: Tuft at one pole (H. pylori)
• Amphitrichous: Flagella at both poles
• Peritrichous: Flagella all around (E. coli, Salmonella, Proteus)
• Atrichous: No flagella (Klebsiella, Shigella)

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SECTION 3: CULTURE MEDIA

Classification of Culture Media

By Consistency

By Purpose

1. Enriched / Non-selective Media

2. Selective Media

3. Differential Media

4. Special/Transport Media

Hemolysis on Blood Agar

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Inoculation Techniques

Streak Plate Method (for isolation of pure culture)

1. Sterilize inoculating loop in flame; cool in non-contaminated area
2. Pick inoculum, streak sector 1 (1/4 plate) with back-and-forth motion
3. Flame loop, rotate plate 90°; streak sector 2 through last 2 streaks of sector 1
4. Repeat for sectors 3 and 4
5. Incubate inverted at 37°C for 18-24 h

Pour Plate Method

• Melt agar to 45-50°C, add inoculum, pour into Petri dish
• Colonies grow WITHIN agar (subsurface) and on surface

Spread Plate Method

• Spread 0.1 mL diluted sample evenly on surface of pre-poured plate with glass spreader (Drigalski spreader/hockey stick)
• All colonies grow on surface - used for colony counting (CFU)

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SECTION 4: STERILIZATION AND DISINFECTION

Definitions (Exam-critical)

Methods of Sterilization

A. Physical Methods

1. MOIST HEAT (Most reliable)

• Q: What is the principle of autoclave? A: Saturated steam under pressure. At 15 psi, steam reaches 121°C (above normal boiling point). Moist heat denatures and coagulates bacterial proteins irreversibly.
• Q: How do you check autoclave efficiency? A: Browne's tube (chemical indicator - green to red); Bowie-Dick tape; Spore strips (Bacillus stearothermophilus - biological indicator, gold standard)
• Q: Why is moist heat better than dry heat? A: Moist heat coagulates proteins at lower temperatures; dry heat oxidizes; proteins coagulate easier in presence of water

2. DRY HEAT

3. RADIATION

4. FILTRATION

• Membrane filters (0.22 μm): Remove bacteria (not viruses); sterilize heat-labile solutions (serum, vitamins, antibiotics, eye drops)
• HEPA filters (0.3 μm): Remove bacteria + fungal spores from air; used in OT, BSC, isolation rooms
• Candle/Seitz filters: Old methods; largely replaced

B. Chemical Methods

• Q: Why 70% alcohol and not 100%? A: Pure alcohol dehydrates bacterial surface too quickly forming a coagulated protein barrier that prevents penetration. 70% (with water) penetrates better and denatures proteins throughout
• Q: What is the gold standard disinfectant? A: Glutaraldehyde 2% - HIGH level disinfectant (kills mycobacteria, spores with prolonged contact), used for endoscopes
• Q: What is ETO used for? A: Sterilization of heat-sensitive, moisture-sensitive medical devices (catheters, cardiac pacemakers, plastic items)

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SECTION 5: ANTIBIOTIC SENSITIVITY TESTING (AST)

Kirby-Bauer Disc Diffusion Method

Principle

Medium Used

• Well-defined composition
• Good batch-to-batch reproducibility
• Low in sulfonamide/tetracycline inhibitors
• Allows most bacteria to grow

Procedure

McFarland Standard

• 0.5 McFarland = turbidity equivalent to ~1.5 × 10^8 bacteria/mL
• Made by mixing BaCl2 + H2SO4 to form BaSO4 precipitate
• Used to standardize inoculum density

Results Interpretation

• Large ZOI = organism is sensitive (drug effective)
• Small/No ZOI = organism is resistant
• Each drug has specific breakpoints (CLSI/EUCAST tables):

MIC (Minimum Inhibitory Concentration)

• Definition: Lowest concentration of antibiotic that inhibits visible bacterial growth
• Methods: Broth microdilution (gold standard), Etest strips
• Disc diffusion gives qualitative result (S/I/R); MIC gives quantitative result
• MBC (Minimum Bactericidal Concentration): Lowest concentration that kills ≥99.9% of bacteria

• Q: Why is Mueller-Hinton agar used for AST? A: Standardized composition, allows reproducible results, low level of sulfonamide inhibitors, supports growth of most pathogens
• Q: What is 0.5 McFarland standard? A: Turbidity standard = 1.5 × 10^8 CFU/mL; standardizes inoculum density for AST
• Q: What is MIC? A: Minimum Inhibitory Concentration = lowest antibiotic concentration that inhibits visible growth in broth dilution test
• Q: Difference between bactericidal and bacteriostatic antibiotics? A: Bactericidal drugs kill (e.g., penicillin, aminoglycosides); bacteriostatic drugs inhibit growth (e.g., tetracyclines, chloramphenicol) - bacteriostatic drugs rely on host immunity to clear organisms

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SECTION 6: ORGANISM IDENTIFICATION - BIOCHEMICAL TESTS

Key Biochemical Tests

Catalase Test

• Purpose: Distinguish Staphylococcus (+ve) from Streptococcus (-ve)
• Reagent: 3% H2O2
• Method: Add H2O2 to colony on glass slide or tube
• Positive: Immediate effervescence (bubbles) - O2 gas produced by catalase enzyme: 2H2O2 → 2H2O + O2
• Negative: No bubbles

Coagulase Test (for Staphylococcus)

• Purpose: Distinguish S. aureus (+ve) from CONS (coagulase-negative Staph)
• Two forms:
  • Slide test (bound coagulase/clumping factor): Emulsify colony in saline, add plasma; positive = clumping in 10 sec
  • Tube test (free coagulase): Mix colony with plasma in tube; incubate 37°C; positive = gel/clot in 1-4 h (gold standard)
• Coagulase converts fibrinogen → fibrin

Oxidase Test

• Purpose: Detect cytochrome C oxidase (present in organisms using O2 as final electron acceptor)
• Reagent: Tetramethyl-p-phenylenediamine (Kovac's oxidase reagent)
• Method: Smear colony onto filter paper soaked with reagent
• Positive: Purple/blue color within 10-30 sec (Pseudomonas, Neisseria, V. cholerae, Campylobacter)
• Negative: No color change (Enterobacteriaceae - E. coli, Klebsiella, Salmonella)

Indole Test

• Purpose: Detect tryptophanase enzyme (breaks tryptophan → indole + pyruvate + ammonia)
• Reagent: Kovac's reagent (p-dimethylaminobenzaldehyde + isoamyl alcohol + HCl)
• Method: Grow organism in tryptophan broth; add Kovac's reagent
• Positive: Pink/red ring at top (E. coli +ve; Klebsiella -ve)

Urease Test

• Purpose: Detect urease enzyme (breaks urea → NH3 + CO2 → alkaline shift)
• Medium: Christensen's urea agar (contains urea + phenol red indicator)
• Positive: Pink/Magenta color (Proteus mirabilis - rapid urease; H. pylori - CLO test; Kryptococcus)
• Negative: Yellow/Orange (no color change)

IMViC Tests (for Enterobacteriaceae identification)

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MBBS University Practical Exam - What to Expect

Spot/Identify Practical (Slide/Plate given)

1. A stained slide - identify organism, stain, results
2. A culture plate - identify medium, colony characteristics, likely organism
3. An instrument/equipment - name, principle, sterilization method

Standard Viva Format at Exam

1. Name of stain
2. Name of organism seen
3. Staining principle
4. Step-by-step procedure
5. Common errors + their effects
6. Diagnostic significance

1. Name the medium
2. Type of medium (selective/differential/etc.)
3. Colony characteristics (size, shape, colour, hemolysis, smell)
4. Likely organism
5. Other organisms that grow/don't grow on this medium

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Quick Revision: Mnemonics

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