Free ASCP MLS Exam Practice Questions Mock Test: Part 18 – Microbiology – Analytic Procedures for Mycobacteriology

• The Runyon classification groups nontuberculous mycobacteria (NTM) into four categories based on:

  1. Pigment production:

     • Photochromogens (Group I): Produce pigment only after light exposure (e.g., M. kansasii).

     • Scotochromogens (Group II): Produce pigment in light or dark (e.g., M. scrofulaceum).

     • Nonchromogens (Group III): No pigment (e.g., M. avium complex).

  2. Growth rate:

     • Rapid growers (Group IV): Visible colonies in ≤7 days (e.g., M. fortuitum).

     • Slow growers (Groups I–III): Require >7 days.

Why Not the Others?

• b) Drug susceptibility → Not part of Runyon’s criteria (though clinically relevant).

• c) Colony morphology → Describes texture (e.g., rough/smooth) but isn’t the basis of classification.

• d) Antigenic structure → Used for serotyping, not Runyon groups.

• Rifampin (Rifampicin) is a first-line antituberculosis drug, part of the standard regimen for treating drug-susceptible tuberculosis (TB). It is typically used in combination with isoniazid (H), pyrazinamide (Z), and ethambutol (E) during the intensive phase (e.g., 2HRZE) and continued with isoniazid and rifampin in the continuation phase (e.g., 4HR) .

Why Not the Others?

• a) Amphotericin B → An antifungal, not used for TB.

• b) Vancomycin → An antibiotic for Gram-positive bacterial infections (e.g., MRSA), not TB.

• d) Ciprofloxacin → A fluoroquinolone, reserved for drug-resistant TB or when first-line drugs are contraindicated

• Class II biosafety cabinets (BSCs) are the standard for processing mycobacterial specimens (e.g., sputum, tissue) because they provide:

  • Protection for the user: HEPA-filtered exhaust prevents aerosol exposure to M. tuberculosis or other pathogens.

  • Protection for the sample: Laminar airflow maintains sterility during culture or staining procedures.

  • Environmental containment: HEPA filters trap infectious particles before air is recirculated or exhausted.

Why Not the Others?

• a) Class I → Only protects the user/environment (no sample protection); used for low-risk work.

• c) Class III → Fully enclosed, gas-tight for maximum containment (e.g., BSL-4 labs); overkill for routine mycobacteriology.

• d) Class IV → Not a recognized BSC class (distractor).

• Photochromogens (e.g., Mycobacterium kansasii) are a group of non-tuberculous mycobacteria (NTM) that produce pigment (carotenoid) only after exposure to light.

  • When grown in the dark, colonies are non-pigmented (buff-colored).

  • When exposed to light (photostimulated), they turn yellow/orange.

• This distinguishes them from:

  • Scotochromogens (pigmented in light & dark, e.g., M. scrofulaceum).

  • Nonchromogens (no pigment, e.g., M. avium complex).

Why not the others?

• a) High CO₂ – Used for some fastidious bacteria, not mycobacterial pigment.

• c) Specific nutrients – Not required for photochromogen pigment.

• d) Low temperature – Affects growth rate, not pigment production.

• Drug susceptibility testing (DST) for Mycobacterium tuberculosis is routinely performed for first-line anti-TB drugs, which include:

  • Rifampin (R)

  • Isoniazid (H)

  • Pyrazinamide (Z)

  • Ethambutol (E)

• These drugs form the backbone of standard TB treatment regimens, and DST ensures effective therapy by detecting resistance early.

Why Not the Others?

• a) Only rifampin → While rifampin resistance is a critical marker (often indicating MDR-TB), DST is not limited to it.

• c) Second-line drugs only → Tested when resistance to first-line drugs is confirmed (e.g., for MDR/XDR-TB).

• d) All antimicrobial agents → Impractical; testing focuses on clinically relevant TB drugs.

• Mycobacterium kansasii is a photochromogen (Runyon Group I) that:

  • Grows at both 30°C and 37°C, unlike M. marinum (optimal at 30°C, poor/no growth at 37°C).

  • Produces bright yellow pigment when exposed to light (non-pigmented in the dark).

• It is a slow grower (colonies in 2–3 weeks) and causes TB-like pulmonary disease or disseminated infections in immunocompromised hosts .

Why Not the Others?

• b) M. marinum → Grows optimally at 28–32°C (fails at 37°C); causes “swimming pool granuloma.”

• c) M. scrofulaceum → A scotochromogen (yellow in light/dark), not photochromogen.

• d) M. gordonae → A scotochromogen (environmental, rarely pathogenic).

• NALC-NaOH (N-acetyl-L-cysteine + sodium hydroxide) is the most common decontamination method for mycobacterial culture (e.g., sputum). It:

  • Liquefies mucus (via NALC, a mucolytic agent).

  • Kills contaminating bacteria/fungi (via NaOH), while sparing hardy mycobacteria.

  • Is used in standard protocols (e.g., CDC, WHO) for concentrating acid-fast bacilli (AFB) before culture/PCR.

Why Not the Others?

• b) HCl-pepsin → Rarely used; less effective for viscous specimens.

• c) Potassium permanganate → A disinfectant, not a decontaminant for lab processing.

• d) Ethanol → Fixes specimens (e.g., for histopathology), but kills mycobacteria—unsuitable for culture.

• Sputum is the most sensitive specimen for detecting Mycobacterium tuberculosis in chronic pulmonary TB, as it directly samples the primary site of infection (lungs) where bacilli are most concentrated .

  • Studies show that sputum culture (e.g., on Lowenstein-Jensen medium or MGIT) has higher recovery rates compared to extrapulmonary specimens like urine or blood .

  • In smear-positive cases, sputum yields >10,000 bacilli/mL, making it optimal for AFB staining and molecular tests like Xpert MTB/RIF .

Limitations of Other Specimens:

• Urine (a): Useful for genitourinary TB but has lower sensitivity due to dilution and intermittent bacillary excretion.

• Blood (c): Rarely positive except in disseminated TB (e.g., advanced HIV).

• Bone marrow (d): Invasive and typically reserved for suspected miliary TB with negative sputum cultures

The Mycobacterium tuberculosis complex (MTBC) includes closely related pathogenic mycobacteria that cause tuberculosis in humans and animals. Key members are:

1. M. tuberculosis (primary human pathogen)

2. M. bovis (causes TB in cattle and humans, often via unpasteurized milk)

3. M. africanum (endemic in West Africa)

4. M. microti (rodent TB)

5. M. canetti (rare, smooth-colony variant)

Why Other Options Are Incorrect:

• a) M. avium → Part of non-tuberculous mycobacteria (NTM); causes pulmonary disease in immunocompromised patients (MAC infection).

• b) M. kansasii → NTM causing lung infections, often in HIV patients.

• d) M. fortuitum → Rapidly growing NTM (skin/soft tissue infections).

• Mycobacterium marinum is a slow-growing, photochromogenic non-tuberculous mycobacterium.

• It causes skin and soft tissue infections (e.g., “fish tank granuloma”) because it prefers cooler temperatures found in peripheral tissues.

• Optimal growth occurs between 28 °C and 32 °C — laboratories typically incubate cultures at 30 °C.

Why not the others?

• (a) 25 °C → Too low; growth is slower.

• (c) 37 °C → More suitable for M. tuberculosis, not M. marinum.

• (d) 42 °C → Too high; used for thermophilic organisms like Pseudomonas aeruginosa.

• Cord formation in Mycobacterium tuberculosis cultures is primarily mediated by trehalose 6,6′-dimycolate (TDM, or “cord factor”), a glycolipid composed of mycolic acids esterified to trehalose .

  • Mycolic acids are long-chain fatty acids that form a waxy outer layer in the mycobacterial cell wall, enabling tight aggregation of bacteria into serpentine cords.

  • TDM’s amphiphilic nature allows it to form rigid monolayers or micelles, driving the side-by-side and end-to-end alignment of bacilli .

Why Not the Others?

• b) Niacin accumulation → Niacin production is a biochemical test for M. tuberculosis identification but unrelated to cord formation.

• c) Lipoarabinomannan (LAM) → A virulence factor that inhibits phagosome-lysosome fusion but does not contribute to cording.

• d) Sulfatides → Sulfolipids involved in immune evasion but not bacterial aggregation

• Rapid-growing mycobacteria (RGM) are defined by their ability to form visible colonies on solid media (e.g., Lowenstein-Jensen or Middlebrook agar) within ≤7 days under optimal conditions.

  • Examples: M. fortuitum, M. chelonae, M. abscessus.

• Slow growers (e.g., M. tuberculosis, M. avium complex) require weeks (≥7 days) for visible growth.

Why Not the Others?

• a) Pigment production → Depends on species (Runyon classification), not growth speed.

• c) Resistance to rifampin → Some rapid growers are susceptible (e.g., M. abscessus can be sensitive).

• d) Ability to grow without oxygen → Mycobacteria are aerobic; growth rate is unrelated to oxygen tolerance.

• Mycobacterium tuberculosis colonies typically exhibit:

  • Rough, dry, and crumbly texture (often described as “cauliflower-like”).

  • Buff (beige/tan) color (non-pigmented, as it is a nonchromogen).

  • Slow growth (3–6 weeks on Löwenstein-Jensen or Middlebrook agar).

Why not the others?

• a) Yellow pigment → Seen in photochromogens (e.g., M. kansasii) or scotochromogens (e.g., M. gordonae).

• b) Smooth texture → More typical of non-tuberculous mycobacteria (NTM) (e.g., M. avium complex).

• d) Mucoid appearance → Suggests NTM like M. abscessus or M. mucogenicum.

• Mycobacterium bovis BCG (Bacille Calmette-Guérin) is an attenuated live strain derived from M. bovis and is primarily used as a vaccine against tuberculosis (TB).

  • It provides partial protection against severe forms of TB (e.g., disseminated TB in children) but is less effective against pulmonary TB in adults .

  • BCG is also used in bladder cancer immunotherapy due to its immunomodulatory effects .

Why Not the Others?

• a) Leprosy treatment → BCG may offer some cross-protection against leprosy but is not a treatment.

• c) MAC infection prevention → No proven efficacy; macrolides (e.g., azithromycin) are used for MAC prophylaxis in AIDS patients .

• d) MDR-TB therapy → BCG is not a treatment for active TB or drug-resistant TB.

• Isoniazid (INH) resistance in M. tuberculosis is most commonly caused by mutations in the katG gene, which encodes catalase-peroxidase.

  • The Ser315Thr mutation (present in ~50–95% of INH-resistant strains) reduces KatG’s ability to activate INH into its bactericidal form .

• Other mechanisms include:

  • inhA promoter mutations (15–20% of resistant strains), which upregulate InhA (enoyl-ACP reductase), the target of activated INH .

  • Less commonly, ahpC or ndh mutations (rare).

Why Not the Others?

• b) rpoB → Confers rifampin resistance (not INH).

• c) gyrA → Associated with fluoroquinolone resistance (e.g., levofloxacin).

• d) inhA → While important, it is secondary to katG in frequency.

• Mycobacterium szulgai is classified as a scotochromogen under the Runyon classification system.

  • Scotochromogens produce pigment regardless of light exposure (yellow/orange in both light and dark conditions).

  • M. szulgai is unique because it exhibits temperature-dependent pigmentation:

    • At 37°C: Acts as a scotochromogen (yellow in light/dark).

    • At 25°C: May show photochromogenic traits (pigments only with light).

Why Not the Others?

• b) Nonchromogen → Incorrect; M. szulgai produces pigment (unlike M. avium complex).

• c) Photochromogen → Only partially correct (at 25°C, not 37°C).

• d) Rapid grower → Incorrect; M. szulgai is a slow grower (visible colonies in 2–4 weeks).

• The PANTA antibiotic mixture (Polymyxin B, Amphotericin B, Nalidixic Acid, Trimethoprim, and Azlocillin) is specifically formulated to prevent contamination in liquid mycobacterial cultures (e.g., MGIT, BACTEC). It targets:

  • Bacteria (Gram-positive/negative via polymyxin B, nalidixic acid, trimethoprim).

  • Fungi (via amphotericin B).

• PANTA is added to Middlebrook 7H9 broth or MGIT tubes to suppress contaminants while sparing mycobacteria .

Why Not the Others?

• a) Malachite green → Used in solid media (e.g., Löwenstein-Jensen) but ineffective in liquid cultures.

• c) Rifampin → An anti-TB drug; would inhibit M. tuberculosis growth if added to culture media.

• d) NaOH → Used for specimen decontamination (e.g., NALC-NaOH method) but not added to culture media (toxic to mycobacteria at high concentrations)

• Cerebrospinal fluid (CSF) is the least likely specimen to require digestion-decontamination because it is:

  • Naturally sterile (no contaminating bacteria/fungi unless due to trauma or contamination during collection).

  • Paucibacillary (low mycobacterial load), making aggressive decontamination (e.g., NALC-NaOH) risky for killing M. tuberculosis bacilli.

• Instead, CSF is typically centrifuged to concentrate bacilli and inoculated directly into culture media (e.g., MGIT, LJ) .

Why Not the Others?

• a) Sputum / d) Bronchial lavage → Require digestion (NALC) and decontamination (NaOH) to liquefy mucus and kill oral flora.

• b) Urine → Often contaminated with genital flora; decontamination (e.g., HCl) is standard unless collected via sterile catheter .

• Mycobacterium marinum is a slow-growing, photochromogenic non-tuberculous mycobacterium.

• It causes skin and soft tissue infections (e.g., “fish tank granuloma”) because it prefers cooler temperatures found in peripheral tissues.

• Optimal growth occurs between 28 °C and 32 °C — laboratories typically incubate cultures at 30 °C.

Why not the others?

• (a) 25 °C → Too low; growth is slower.

• (c) 37 °C → More suitable for M. tuberculosis, not M. marinum.

• (d) 42 °C → Too high; used for thermophilic organisms like Pseudomonas aeruginosa.

• Auramine-rhodamine staining is the most sensitive method for detecting low numbers of acid-fast bacilli (AFB) due to its fluorescent properties, which allow:

  • Enhanced visibility: Bacilli emit bright yellow-green fluorescence against a dark background, making even sparse organisms detectable.

  • Lower magnification screening: Can be scanned at 20x or 40x magnification (vs. 100x oil immersion required for Ziehl-Neelsen/Kinyoun), enabling faster examination of larger sample areas.

• Studies show it improves sensitivity by 10–30% over Ziehl-Neelsen for paucibacillary specimens (e.g., early TB, HIV-coinfection) .

Why Not the Others?

• a) Ziehl-Neelsen / b) Kinyoun → Less sensitive due to reliance on light microscopy and small field of view at 100x oil immersion.

• d) Gram stain → Cannot reliably detect AFB (mycobacteria stain poorly or appear “ghost-like”).

• Auramine-rhodamine staining is a fluorescent acid-fast staining method that:

  • Detects mycobacteria more sensitively than Ziehl-Neelsen (Kinyoun) under a fluorescence microscope, especially in paucibacillary specimens (e.g., early TB or HIV-coinfected patients).

  • Allows faster screening at lower magnification (e.g., 20x or 40x vs. 100x oil immersion in ZN).

Why Not the Others?

• a) Is cheaper → False; fluorescent staining is typically more expensive due to microscope requirements.

• b) Requires no microscope → False; it requires a fluorescence microscope.

• d) Works on fixed specimens → True, but Ziehl-Neelsen also works on fixed specimens, so this is not a unique advantage.

• Sodium hydroxide (NaOH) in the NALC-NaOH (N-acetyl-L-cysteine + NaOH) method serves as a decontaminating agent to:

  • Kill contaminating bacteria and fungi in sputum or other specimens, which would otherwise overgrow mycobacteria during prolonged culture incubation.

  • Reduce sample viscosity (though this is primarily the role of NALC).

• NaOH’s alkaline action (typically 1–2% concentration) is harsh but selective:

  • Most non-mycobacterial organisms are killed, while mycobacteria survive due to their hardy, lipid-rich cell walls.

Why Not the Others?

• a) Liquefy mucus → Achieved by NALC (a mucolytic agent), not NaOH.

• c) Enhance growth of mycobacteria → NaOH does not promote growth; it merely removes competitors.

• d) Stain AFB → Staining requires carbol fuchsin or fluorescent dyes (e.g., auramine), not NaOH.

• Lowenstein-Jensen (L-J) agar is a selective medium specifically designed for the cultivation of mycobacteria, including Mycobacterium tuberculosis.

• It contains malachite green to inhibit other bacteria and provides essential nutrients (like egg yolk and glycerol) that support the slow growth of mycobacteria.

• Blood agar (a) and chocolate agar (d) are used for fast-growing bacteria, not acid-fast bacilli.

• MacConkey agar (c) is used for Gram-negative enteric bacteria, not mycobacteria.

• Digestion–decontamination is needed for specimens that are likely contaminated with normal flora and contain mucus or debris — common in respiratory samples.

• Bronchial washings, like sputum, often have:

  • Mucus → requires N-acetyl-L-cysteine (NALC) to liquefy it.

  • Mixed bacteria → requires NaOH to kill contaminants while preserving mycobacteria.

• Sterile body fluids (like CSF, synovial fluid, pleural fluid) generally do not require this process — they can be directly centrifuged and cultured.

Why not the others?

• (a) CSF → Usually sterile; direct processing is preferred to avoid loss of viable mycobacteria.

• (c) Synovial fluid → Sterile joint fluid; no decontamination needed.

• (d) Pleural fluid → Sterile unless secondarily infected; processed without harsh decontamination.

• Mycobacterium ulcerans is the causative agent of Buruli ulcer, a necrotizing skin disease characterized by painless ulcers, subcutaneous tissue destruction, and immunosuppression due to the toxin mycolactone.

• It is the third most common mycobacterial disease in immunocompetent humans after tuberculosis (caused by M. tuberculosis) and leprosy (caused by M. leprae).

Why Not the Others?

• a) Pulmonary TB → Caused by M. tuberculosis, not M. ulcerans.

• c) Leprosy → Caused by M. leprae.

• d) Swimming pool granuloma → Caused by M. marinum, a relative of M. ulcerans but distinct in pathology and clinical presentation

• The Ziehl-Neelsen (ZN) stain is the gold standard for detecting acid-fast bacilli (AFB), including Mycobacterium tuberculosis.

• It uses carbol fuchsin (red dye) with heat to penetrate the waxy mycolic acid in the bacterial cell wall, followed by acid-alcohol decolorization (which AFB resist, retaining the red color).

• Gram stain (a) does not work well for mycobacteria due to their lipid-rich cell wall.

• Methylene blue (c) is a simple stain, not specific for AFB.

• Calcofluor white (d) is used for detecting fungi, not mycobacteria.

• Lowenstein-Jensen (LJ) medium is a solid culture medium used to isolate Mycobacterium tuberculosis and other mycobacteria. Its key components include:

  1. Egg base: Provides lipids and proteins that support mycobacterial growth.

  2. Malachite green: Inhibits the growth of contaminating bacteria/fungi while allowing mycobacteria to grow.

  3. Glycerol (in some formulations): Enhances growth of M. tuberculosis.

Why Not the Others?

• a) Charcoal and yeast extract → Used in Middlebrook 7H10/7H11 agar, not LJ medium.

• c) Blood and antibiotics → Used in general bacteriology media (e.g., blood agar), not LJ.

• d) Sucrose and agar → Components of MacConkey agar, not LJ.

• Mycobacterium marinum is the causative agent of “swimming pool granuloma” (also called aquarium granuloma or fish tank granuloma), a chronic skin infection acquired through exposure to contaminated water (e.g., swimming pools, aquariums, or fish handling) via breaks in the skin .

Why Not the Others?

• b) M. gordonae → An environmental mycobacterium rarely pathogenic in humans.

• c) M. kansasii → Causes pulmonary infections, not typically skin granulomas.

• d) M. ulcerans → Causes Buruli ulcer, a necrotizing skin disease unrelated to water exposure

• Formalin (formaldehyde solution) can destroy acid-fastness by cross-linking and altering the mycolic acids in the mycobacterial cell wall, which reduces staining affinity in acid-fast stains.

• This can lead to false-negative results in acid-fast staining if specimens are fixed in formalin before staining.

• Therefore, for acid-fast staining, heat-fixed smears without prior formalin fixation are preferred.

Why not the others?

• Methanol, acetone, ethanol → Commonly used as fixatives or in staining procedures but do not significantly affect acid-fastness like formalin does.

• Biological Safety Cabinet (BSC) Class II is essential when processing specimens for mycobacterial culture to:

  • Prevent aerosol generation (a major route of M. tuberculosis transmission).

  • Protect lab personnel from inhaling infectious droplets during procedures like homogenization or centrifugation.

Why Other Options Are Incorrect:

• a) Use of open bench tops → High risk of aerosol exposure; strictly prohibited.

• c) Incubation at room temperature → Mycobacteria require 37°C (or 30°C for M. marinum); room temperature inhibits growth.

• d) Centrifugation without a sealed lid → Aerosols escape; must use sealed rotors or safety cups.

• Mycobacterium gordonae is commonly called the “tap water bacillus” because it is frequently isolated from water sources (e.g., tap water, showers, and soil).

• It is a non-pathogenic or rarely pathogenic mycobacterium, often considered a contaminant in clinical specimens.

Why Not the Others?

• b) Fish tank bacillus → Refers to M. marinum (causes “swimming pool granuloma”).

• c) Buruli ulcer bacillus → Refers to M. ulcerans.

• d) Cow bacillus → Refers to M. bovis.

• Acid-fastness (resistance to decolorization by acid-alcohol in staining procedures like Ziehl-Neelsen or Kinyoun) is a hallmark feature of mycobacteria, primarily due to their unique cell wall composition:

  • Mycolic acids: Long-chain fatty acids that form a waxy, hydrophobic layer, making the cell wall impermeable to routine stains and contributing to acid-fastness.

  • This layer also confers resistance to disinfectants and antibiotics.

Why Not the Others?

• a) Peptidoglycan → Provides structural support but does not confer acid-fastness.

• c) Capsular polysaccharides → Found in some mycobacteria (e.g., M. tuberculosis) but not responsible for acid-fast staining.

• d) Porin proteins → Facilitate nutrient uptake but play no role in acid-fast properties.

• Nocardia species are:

  • Gram-positive

  • Branching filamentous rods

  • Partially acid-fast (due to mycolic acids in their cell wall; detected with a modified acid-fast stain such as Kinyoun using weaker decolorizer).

• They are found in soil and can cause pulmonary, cutaneous, or disseminated infections — pulmonary nocardiosis often mimics TB.

• A bronchial washing growing such an organism strongly suggests Nocardia.

Why not the others?

• (a) Actinomyces → Branching Gram-positive rods but not acid-fast; anaerobic, found in oral flora.

• (c) Streptomyces → Aerobic, branching Gram-positive rods, not acid-fast, mainly environmental.

• (d) Corynebacterium → Gram-positive rods, non-branching, not acid-fast.

• N-acetyl-L-cysteine (NALC) is a mucolytic agent that breaks down disulfide bonds in sputum mucus, reducing viscosity and homogenizing the sample for better processing.

• This allows for:

  • More effective decontamination (e.g., with NaOH or Na₃PO₄).

  • Improved centrifugation and mycobacterial recovery in culture.

Why Other Options Are Incorrect?

• a) Inhibit fungal growth → NALC has no antifungal properties (NaOH or other agents handle this).

• b) Neutralize alkalinity → NALC does not adjust pH (NaOH remains active for decontamination).

• d) Kill contaminants → NALC lacks bactericidal effects (NaOH is the primary decontaminant).

• The catalase test in mycobacteriology helps distinguish isoniazid (INH)-resistant M. tuberculosis strains from susceptible ones.

  • Heat-labile catalase: INH-resistant strains (especially those with katG mutations) often lose catalase activity when heated to 68°C, while susceptible strains retain it.

  • Heat-stable catalase: Most non-tuberculous mycobacteria (NTM) and some INH-resistant strains (e.g., with inhA mutations) remain catalase-positive after heating.

Why Not the Others?

• a) Drug resistance → The catalase test only correlates with INH resistance, not other drugs.

• c) Presence of pigment → Determined by light exposure (Runyon classification), not catalase.

• d) Growth rate → Assessed via culture timing, not enzymatic tests.

• The Kinyoun method is a cold acid-fast stain (no heat required) used to detect acid-fast organisms such as Mycobacterium species.

• Primary stain: Carbol fuchsin — penetrates the waxy, mycolic acid–rich cell wall of acid-fast bacteria.

• Decolorizer: Acid-alcohol — removes stain from non–acid-fast cells.

• Counterstain: Methylene blue (or sometimes brilliant green) — stains the background and non–acid-fast organisms.

• Acid-fast organisms appear red, while non–acid-fast organisms appear blue/green.

Why not the others?

• (a) Crystal violet → Used in Gram staining.

• (c) Methylene blue → Used as the counterstain, not the primary stain.

• (d) Malachite green → Used in spore staining or as an optional counterstain in acid-fast stains.

• “Scanty” is a standardized reporting term for acid-fast bacilli (AFB) smears, indicating very low bacterial load. It is defined as:

  • 1–9 AFB observed per 100 microscopic fields (using 100x oil immersion).

  • This classification helps clinicians interpret the likelihood of true infection vs. contamination (e.g., in paucibacillary TB or early disease).

Why Not the Others?

• b) 10–99 AFB per field → Reported as “1+” (moderate).

• c) >100 AFB per field → Reported as “2+” (heavy) or “3+” (very heavy).

• d) No AFB seen → Reported as “negative”.

• Mycobacterium avium-intracellulare complex (MAC) consists of multiple closely related species (e.g., M. avium, M. intracellulare, M. chimaera).

• Biochemical tests (e.g., niacin test, growth rate, pigmentation) are unreliable for differentiation because MAC members share similar phenotypic traits.

• Molecular methods (e.g., PCR, gene sequencing [16S rRNA, hsp65, rpoB], MALDI-TOF MS) provide definitive speciation by detecting genetic differences.

Why Not the Others?

• a) Niacin test → Used to distinguish M. tuberculosis (positive) from most NTMs (negative), but MAC is niacin-negative.

• b) Growth rate → MAC grows slowly (similar to other NTMs), so this does not differentiate within the complex.

• d) Pigmentation → MAC is typically nonchromogenic (buff-colored), like many other NTMs.

• Mycobacterium ulcerans is the causative agent of Buruli ulcer, a chronic, necrotizing skin infection characterized by painless ulcers.

• It produces a toxin called mycolactone, which causes tissue destruction and suppresses the immune response.

• Buruli ulcer is most common in tropical and subtropical regions, especially in West Africa.

Why not the others?

• M. avium → Causes pulmonary and disseminated infections, especially in immunocompromised.

• M. marinum → Causes “swimming pool granuloma” (skin infections after water exposure).

• M. fortuitum → Rapid-growing NTM causing skin and soft tissue infections, but not Buruli ulcer.

• The niacin test detects the accumulation of free niacin (nicotinic acid) in the culture medium.

• Mycobacterium tuberculosis is niacin positive, meaning it produces and accumulates niacin, which can be detected by this biochemical test.

• This helps differentiate it from many other mycobacteria, which are typically niacin negative.

Why not the others?

• M. kansasii → Niacin negative

• M. fortuitum → Niacin negative

• M. avium → Niacin negative

• Mycolic acids, long-chain fatty acids unique to mycobacteria, are the primary determinant of acid-fastness. They form a waxy, hydrophobic layer that:

  • Retains carbol fuchsin dye during acid-alcohol decolorization (e.g., in Ziehl-Neelsen staining).

  • Blocks penetration of aqueous stains (e.g., Gram stain), making mycobacteria appear “Gram-neutral.”

• Mycolic acids are covalently linked to arabinogalactan (c), which anchors them to the peptidoglycan (a) layer, but these components alone do not confer acid-fastness.

Why Not the Others?

• a) Peptidoglycan → Provides structural support but lacks lipid content for acid-fastness.

• c) Arabinogalactan → Connects mycolic acids to peptidoglycan but is not directly responsible for staining properties.

• d) Lipoarabinomannan (LAM) → A virulence factor (modulates host immunity) but does not contribute to acid-fastness.

• Mycobacterium kansasii is classified as a photochromogen in the Runyon classification of nontuberculous mycobacteria (NTM). This means it produces a yellow pigment when exposed to light (photostimulation) but remains non-pigmented when grown in the dark .

• This property distinguishes it from:

  • Scotochromogens (e.g., M. scrofulaceum), which produce pigment in both light and dark conditions.

  • Nonchromogens (e.g., M. avium complex), which lack pigment entirely.

  • Rapid growers (e.g., M. fortuitum), which form colonies within 7 days, unlike M. kansasii (a slow grower)

• Mycobacterium haemophilum is a fastidious mycobacterium that requires hemin (an iron-containing porphyrin) supplementation for growth.

• It typically grows best at lower temperatures (~30–32°C) on media supplemented with hemin or blood.

• This requirement distinguishes it from many other mycobacteria.

Why not the others?

• Mycobactin J → Required by Mycobacterium avium subspecies paratuberculosis (MAP).

• Iron salts → General nutrient but not specifically required like hemin for M. haemophilum.

• Egg yolk → Used in media like Lowenstein-Jensen but not a specific growth requirement here.

• Photochromogens (Group I in the Runyon classification) produce a yellow or orange-yellow pigment when exposed to light. This is due to the synthesis of carotenoid pigments triggered by light exposure.

  • Example: Mycobacterium kansasii colonies appear non-pigmented (buff-colored) in the dark but turn bright yellow after light exposure .

Why Not the Others?

• a) Red → Seen in some Serratia or Rhodococcus spp., not mycobacteria.

• c) Blue → Rare in bacteria; not associated with photochromogens.

• d) Green → Unrelated to mycobacterial pigmentation.

• In the Ziehl-Neelsen (ZN) staining method for acid-fast bacilli (AFB), acid-alcohol (3% HCl in 95% ethanol) serves as the decolorizer. Its roles are:

  • Removes carbol fuchsin from non-acid-fast cells (e.g., human cells, contaminating bacteria).

  • Retains carbol fuchsin in acid-fast bacilli (e.g., M. tuberculosis) due to their mycolic acid-rich cell walls.

• This step is critical for contrast: Only AFB remain red, while non-AFB take up the counterstain (methylene blue).

Why Not the Others?

• a) Primary stain → Carbol fuchsin is the primary stain.

• b) Counterstain → Methylene blue or malachite green is the counterstain.

• d) Mordant → Heat acts as the mordant in ZN staining (enhances carbol fuchsin penetration).

• Mycobacterium avium complex (MAC) colonies are typically smooth and non-pigmented (nonchromogenic).

• MAC organisms grow slowly and are often isolated from environmental sources and immunocompromised patients.

• They lack the characteristic pigments seen in photochromogens or scotochromogens.

Why not the others?

• a) Rapid growth → MAC is slow-growing, not rapid.

• c) Rough, pigmented → Typical of some other mycobacteria like M. kansasii.

• d) Mucoid, yellow → Not characteristic of MAC colonies

• Mycobacterium marinum is a slow-growing, photochromogenic mycobacterium that thrives at lower temperatures (25–32°C) and struggles to grow at human core body temperature (37°C) .

• Its optimal growth temperature is 30–32°C, which explains why infections in humans are typically limited to cooler superficial tissues like the skin and extremities .

• Incubation at 37°C (human body temperature) inhibits its growth, distinguishing it from M. tuberculosis .

• Temperatures outside this range (e.g., 25°C or 35°C) may support growth but are suboptimal, while 42°C is too high for survival

• QuantiFERON-TB Gold (QFT) is an interferon-gamma release assay (IGRA) that detects cell-mediated immunity to M. tuberculosis.

• It measures T-cell release of IFN-γ in response to TB-specific antigens (ESAT-6, CFP-10, and TB7.7), which are absent in BCG and most NTMs.

Why Not the Others?

• a) AFB in sputum → Detected by microscopy/culture, not QFT.

• b) Antibodies to M. tuberculosis → Serologic tests are unreliable for TB diagnosis.

• d) Bacterial DNA in CSF → Requires PCR (e.g., Xpert MTB/RIF).

• Malachite green in Löwenstein-Jensen (LJ) medium acts as a selective agent to suppress the growth of contaminating bacteria and fungi, while allowing mycobacteria to grow.

• This is critical because:

  • Mycobacterial cultures require weeks of incubation, during which contaminants could overgrow.

  • The dye’s bacteriostatic/fungistatic properties target fast-growing organisms but do not affect hardy mycobacteria.

Why Not the Others?

• a) Enhance pigment production → Pigmentation depends on mycobacterial species (e.g., photochromogens), not malachite green.

• c) Provide a pH indicator → LJ medium lacks pH indicators; malachite green is not pH-sensitive.

• d) Improve colony morphology → Colony morphology is intrinsic to mycobacteria (e.g., rough vs. smooth), unaffected by the dye.

• Leprosy is caused by Mycobacterium leprae.

• M. leprae cannot be cultured in artificial media (including LJ medium) — it grows only in living hosts (e.g., mouse footpads, armadillos).

• Diagnosis relies on clinical signs (hypopigmented, anesthetic skin lesions, thickened nerves) plus laboratory confirmation using:

  • Skin slit smear or skin biopsy stained with Ziehl–Neelsen or Fite–Faraco stain to detect acid-fast bacilli.

Why not the others?

• (a) Culture on LJ medium → Not possible; M. leprae is non-culturable in vitro.

• (b) Urine antigen test → No such standard test for leprosy.

• (d) Sputum PCR → Useful for TB, not leprosy (unless respiratory involvement, which is rare).

• The Ziehl-Neelsen (ZN) staining method uses phenol (in the carbol fuchsin solution) to enhance dye penetration into the waxy, mycolic acid-rich cell walls of mycobacteria.

  • Phenol acts as a mordant, breaking down the lipid barrier and allowing carbol fuchsin to bind tightly to the cell wall.

  • Heat fixation further aids penetration by softening the cell wall.

Why Not the Others?

• b) Gram stain → Uses crystal violet and iodine, not phenol; mycobacteria stain poorly or appear “ghost-like.”

• c) Acridine orange → A fluorescent nucleic acid stain; does not require phenol.

• d) Wright-Giemsa → Used for blood smears; stains cellular components, not acid-fast bacilli.

• The MGIT (Mycobacteria Growth Indicator Tube) system detects mycobacterial growth through:

  • Oxygen consumption: As mycobacteria grow, they metabolize oxygen in the liquid medium.

  • Fluorescence quenching: The tube contains a silicon-based sensor embedded with a fluorescent dye that is quenched (stops glowing) when oxygen is depleted.

  • A UV light scanner detects this loss of fluorescence, signaling positive growth.

Why Not the Others?

• a) Turbidity measurement → Used in conventional broth systems (e.g., BACTEC 460), not MGIT.

• b) CO₂ production → Detected by systems like BACTEC 12B (radiolabeled CO₂), not MGIT.

• d) pH change → Not part of MGIT’s detection mechanism.

• IS6110 is the most widely targeted gene in nucleic acid amplification tests (NAATs) for M. tuberculosis complex (MTBC) because:

  • It is a highly repetitive insertion sequence (present in 5–20 copies per genome in most MTBC strains), enhancing test sensitivity.

  • It is specific to MTBC, absent in non-tuberculous mycobacteria (NTM).

• Examples of tests using IS6110:

  • Xpert MTB/RIF Ultra (though it primarily targets rpoB for rifampin resistance).

  • Commercial PCR assays (e.g., Cobas TaqMan MTB).

Why Not the Others?

• a) katG / d) inhA → Targeted for isoniazid resistance testing, not primary MTBC detection.

• b) rpoB → Used to detect rifampin resistance (e.g., in Xpert MTB/RIF) but less sensitive than IS6110 for MTBC identification alone.

• Low bacterial load is the most common cause of false-negative AFB smears, as microscopy requires ≥5,000–10,000 bacilli/mL of sputum for reliable detection. In paucibacillary samples (e.g., early TB, HIV coinfection, or extrapulmonary TB), bacilli may be present but fall below this threshold, leading to missed diagnoses .

• Studies show false-negative rates are higher for “scanty” (1–9 AFB/100 fields) or trace-positive smears compared to strongly positive smears (e.g., 11% vs. 4% in one EQA analysis) .

Why Not the Others?

• a) Poor staining technique → While suboptimal staining (e.g., inadequate heating in Ziehl-Neelsen) can cause false negatives, it is less frequent than low bacterial load.

• c) Excessive heat during staining → Rare; typically causes over-decolorization (false positives more likely).

• d) Use of auramine stain → Auramine-rhodamine staining is more sensitive than Ziehl-Neelsen for low-burden samples, reducing false negatives

• The niacin test detects free niacin (nicotinic acid) that accumulates during the growth of certain mycobacteria.

• M. tuberculosis produces and accumulates large amounts of niacin, giving a positive niacin test.

• This test is useful for differentiating M. tuberculosis from most other mycobacteria, which are niacin-negative.

Why not the others?

• (a) M. avium → Niacin-negative.

• (b) M. fortuitum → Niacin-negative, rapid grower.

• (d) M. kansasii → Niacin-negative, photochromogen.

• BCG (Bacille Calmette–Guérin) vaccine is a live attenuated strain of Mycobacterium bovis.

• Developed by Albert Calmette and Camille Guérin in 1921 after 230 serial subcultures over 13 years, which reduced its virulence while retaining immunogenicity.

• It is used worldwide for prevention of severe forms of TB (especially TB meningitis and miliary TB in children), though it is less effective at preventing pulmonary TB in adults.

Why not the others?

• (a) M. tuberculosis → The primary cause of human TB, but not the source of BCG.

• (c) M. africanum → A member of the MTBC, mostly found in West Africa.

• (d) M. microti → Rodent pathogen, not used for vaccine production.

• Mycobacterium xenopi is a thermophilic mycobacterium, meaning it thrives at higher temperatures. Its optimal growth temperature is 42–45°C, which distinguishes it from most other nontuberculous mycobacteria (NTM) that prefer 37°C.

• This adaptation allows M. xenopi to colonize hot-water systems (e.g., hospital plumbing, water heaters), where it survives despite standard disinfection measures.

• While it can grow at 37°C (typical lab conditions), growth is slower and less robust compared to 42°C.

Why Not the Others?

• a) 25°C / b) 30°C → Too low; M. xenopi grows poorly or not at all at these temperatures.

• c) 37°C → Supports growth but is suboptimal; incubation at 37°C may delay culture detection (6–8 weeks vs. faster growth at 42°C)

• Scotochromogens are a group of nontuberculous mycobacteria (NTM) that produce pigment (yellow/orange) regardless of light exposure. This distinguishes them from:

  • Photochromogens (e.g., M. kansasii), which pigment only after light exposure.

  • Nonchromogens (e.g., M. avium complex), which never produce pigment.

• Examples of scotochromogens:

  • M. scrofulaceum (causes cervical lymphadenitis).

  • M. gordonae (environmental, rarely pathogenic).

Why Not the Others?

• a) Only in the presence of light → Describes photochromogens, not scotochromogens.

• c) Never → Describes nonchromogens.

• d) Only at low temperature → Incorrect; pigment production is temperature-independent.

• The MGIT (Mycobacteria Growth Indicator Tube) system detects mycobacterial growth through oxygen depletion:

  1. Mycobacteria consume oxygen as they grow, creating a low-oxygen environment in the tube.

  2. A silicon-based sensor at the tube’s base contains a fluorescent dye that glows under UV light when oxygen is present.

  3. As oxygen is depleted, the dye’s fluorescence increases (no longer quenched by oxygen), signaling positive growth .

Why Not the Others?

• b) pH decreases → Not measured in MGIT (though some cultures acidify media).

• c) CO₂ levels rise → Detected in other systems (e.g., BACTEC 460), not MGIT.

• d) Turbidity exceeds threshold → MGIT uses fluorescence, not turbidity.

• In AFB-smear positive respiratory specimens, the fastest way to confirm Mycobacterium tuberculosis complex (MTBC) is nucleic acid amplification testing (NAAT) or other molecular assays.

• These tests (e.g., GeneXpert MTB/RIF) can:

  • Detect MTBC DNA directly from the specimen.

  • Provide results within 24 hours (often in under 2 hours).

  • Identify rifampicin resistance at the same time.

Why not the others?

• (a) Culture on LJ medium → Takes 3–8 weeks for results.

• (b) Niacin accumulation → Requires cultured organisms; cannot be done directly on the specimen.

• (d) Catalase test → A biochemical test for identification, performed after culture growth.

• Mycobacterium tuberculosis is an airborne pathogen requiring Biosafety Level 3 (BSL-3) precautions during specimen handling.

• Processing activities like vortexing, pipetting, or centrifuging can generate infectious aerosols.

• All specimen manipulations must be performed in a Class II Biological Safety Cabinet (BSC) with proper PPE (gloves, lab coat/gown, N95 or higher respirator).

• This prevents laboratory-acquired TB infections.

Why not the others?

• (a) Open-bench processing → Unsafe for airborne pathogens.

• (c) UV light exposure only → Can be used for surface disinfection but does not protect during active handling.

• (d) Chemical fume hood → Designed for chemical vapors, not infectious aerosols.

• Buffy coat (the leukocyte-rich layer obtained after centrifuging whole blood) is the optimal specimen for mycobacterial blood cultures, especially for diagnosing disseminated infections (e.g., M. avium complex in HIV/AIDS).

  • Mycobacteria are intracellular pathogens that reside within macrophages/monocytes, which concentrate in the buffy coat.

  • This increases the yield compared to whole blood or plasma.

Why Not the Others?

• a) Serum/ b) Plasma → Lack host cells, reducing sensitivity.

• c) Whole blood → Less sensitive than buffy coat due to dilution effects.

• In mycobacterial culture, respiratory specimens (like sputum) often contain mucus that traps bacteria and debris.

• N-acetyl-L-cysteine (NALC) is a mucolytic agent that breaks disulfide bonds in mucus glycoproteins, making the specimen less viscous.

• This liquefaction allows the mycobacteria to be released from mucus, improving contact with the decontaminating agent and increasing recovery in culture.

Why not the others?

• (a) Inhibit fungal growth → That’s the role of antifungal agents like cycloheximide.

• (c) Neutralize sodium hydroxide → Done by buffer solution, not NALC.

• (d) Inhibit bacterial growth → Done by NaOH, not NALC.

• Auramine-rhodamine staining is a fluorescent acid-fast staining method widely used for detecting mycobacteria (e.g., M. tuberculosis) in clinical specimens. The dye combination includes:

  • Auramine O: A fluorescent dye that binds to mycolic acids in the mycobacterial cell wall, making the bacilli appear bright yellow-green under a fluorescence microscope.

  • Rhodamine B: A counterstain that enhances contrast by staining background material (e.g., debris) red-orange, improving visibility of the fluorescent bacilli.

Why Not the Others?

• b) Crystal violet and safranin → Used in Gram staining, not acid-fast fluorescence.

• c) Methylene blue and malachite green → Methylene blue is a simple stain; malachite green is used in Löwenstein-Jensen media, not fluorescence.

• d) Acridine orange and ethidium bromide → Used for nucleic acid staining (e.g., in molecular biology), not mycobacteria.

• Non-tuberculous mycobacteria (NTM) are ubiquitous in the environment (soil, water) and generally not transmitted person-to-person.

• While they can colonize healthy individuals without causing disease, they primarily cause infections in immunocompromised hosts (e.g., HIV/AIDS, cystic fibrosis, COPD) or those with structural lung disease—making them opportunistic pathogens.

Why Not the Others?

• a) Always pathogenic → False; NTM can colonize without disease.

• b) Strictly environmental → False; they originate from the environment but can infect humans/animals.

• d) Only zoonotic → False; most NTM infections are environmentally acquired, though some (e.g., M. bovis) are zoonotic.

• Mycobacterium tuberculosis is a niacin-positive bacterium, meaning it produces and accumulates niacin (nicotinic acid) as a metabolic byproduct. This is a key biochemical test used to differentiate it from other mycobacteria.

• a) Rapid growth within 24 hours – Incorrect. M. tuberculosis is a slow-growing bacterium, typically taking 2–6 weeks to form colonies on solid media.

• c) Pigmented colonies – Incorrect. M. tuberculosis forms non-pigmented (buff-colored) colonies, unlike photochromogenic or scotochromogenic mycobacteria (e.g., M. kansasii, M. scrofulaceum).

• d) Motility at 25°C – Incorrect. Mycobacteria are non-motile at any temperature.

• Mycobacterium avium complex (MAC), particularly M. avium and M. intracellulare, is the most common cause of disseminated mycobacterial infections in AIDS patients with CD4 counts <50 cells/μL.

  • Disseminated MAC presents with fever, weight loss, anemia, and hepatosplenomegaly, often with high bacillary loads in blood and bone marrow .

  • MAC is acquired via environmental exposure (e.g., water, soil) and colonizes the GI tract before disseminating .

Why Not the Others?

• a) M. tuberculosis → Causes pulmonary or extrapulmonary TB in HIV/AIDS but is less likely to disseminate widely unless CD4 is very low.

• b) M. kansasii → Primarily causes pulmonary disease (similar to TB), rarely disseminates.

• d) M. gordonae → A non-pathogenic contaminant; rarely causes disease even in immunocompromised hosts.

• Liquid media (e.g., MGIT – Mycobacteria Growth Indicator Tube) detect mycobacterial growth faster (typically 1–3 weeks) compared to solid media (e.g., Löwenstein-Jensen, 3–8 weeks) due to:

  • Better nutrient availability.

  • Automated detection of metabolic activity (oxygen quenching in MGIT).

• Why not the others?

  • a) Slower than solid media → False; liquid media are significantly faster.

  • c) Different pigmentation → Pigment rules (Runyon classification) apply to both liquid/solid media.

  • d) Only at 42°C → False; most mycobacteria grow at 35–37°C (some NTMs tolerate higher temps).

• Rapidly growing mycobacteria (RGM), such as Mycobacterium fortuitum, M. chelonae, and M. abscessus, form visible colonies on solid media within 7 days of incubation.

• This definition is part of the Runyon classification of non-tuberculous mycobacteria (NTM).

Why not the others?

• (a) 24 hours → Too fast; typical for many common bacteria but not mycobacteria.

• (b) 3 days → Some colonies may appear, but the official classification uses ≤7 days.

• (d) 14 days → Falls into the “slow growers” category (e.g., M. tuberculosis).

• While Mycobacterium avium-intracellulare complex (MAC) is the most common cause of nontuberculous mycobacterial (NTM) cervical lymphadenitis in children globally, among the options provided, M. scrofulaceum is historically significant and regionally prevalent .

• M. scrofulaceum is a scotochromogen (produces pigment in light/dark) and was frequently isolated in past decades, particularly in the southern U.S. and parts of Europe, before MAC became dominant .

Why Not the Others?

• a) M. kansasii → Primarily causes pulmonary disease in adults; rare in pediatric lymphadenitis .

• c) M. marinum → Causes skin granulomas (“swimming pool granuloma”) but not lymphadenitis.

• d) M. xenopi → Associated with pulmonary infections in immunocompromised adults; very rare in children .

• Kinyoun’s acid-fast staining (a cold Ziehl-Neelsen modification) uses carbol fuchsin as the primary stain to detect acid-fast bacteria (e.g., Mycobacterium tuberculosis).

  • Unlike Ziehl-Neelsen, Kinyoun’s method does not require heating (phenol in carbol fuchsin enhances penetration).

• Other options:

  • Methylene blue (a): Counterstain in acid-fast staining.

  • Auramine O (c): Used in fluorescent acid-fast stains (e.g., Truant method).

  • Crystal violet (d): Used in Gram staining, not acid-fast.

• The Xpert MTB/RIF assay is a rapid molecular test that detects Mycobacterium tuberculosis DNA and mutations in the rpoB gene associated with rifampin resistance.

• It provides results within 2 hours, allowing quick diagnosis and initiation of appropriate therapy.

• This is much faster than culture methods, which take weeks.

Why not the others?

• a) Culture on LJ medium → Takes 3–8 weeks for growth and susceptibility testing.

• c) Niacin accumulation test → Biochemical test for species identification, not drug resistance.

• d) Catalase test → Biochemical test, no relevance to rifampin resistance.

• Middlebrook 7H10 and 7H11 are agar-based media used for culturing mycobacteria, but they differ in composition:

  • 7H11 contains casein hydrolysate, which enhances recovery of fastidious mycobacteria (e.g., M. tuberculosis from sputum or drug-treated patients).

  • 7H10 lacks this enrichment, making it less supportive for damaged or slow-growing bacilli.

• Both media share:

  • OADC enrichment (oleic acid, albumin, dextrose, catalase) added before use.

  • Malachite green (inhibits contaminants).

  • Similar incubation conditions (35–37°C, 5–10% CO₂).

Why Not the Others?

• a) Incubation temperature → Identical for both media.

• b) Antibiotic content → Neither contains antibiotics unless supplemented (e.g., PANTA for liquid media).

• d) Malachite green concentration → Comparable in both (~0.25 g/L).

Photochromogens are a group of nontuberculous mycobacteria (NTM) that produce pigment (yellow/orange) only when exposed to light (photoreactive carotenoids). Key features:

1. Mycobacterium kansasii:

   • Colonies are nonpigmented in the dark but turn bright yellow/orange after light exposure.

   • Causes chronic pulmonary disease resembling tuberculosis (especially in immunocompromised patients).

2. Other Options:

   • a) M. tuberculosis → Nonchromogen (no pigment, regardless of light).

   • c) M. avium → Nonchromogen (part of the M. avium-intracellulare complex/MAC).

   • d) M. fortuitum → Rapid grower (pigment varies but not light-dependent).

• Mycobacterium tuberculosis is classified as a Risk Group 3 pathogen, requiring Biosafety Level 3 (BSL-3) containment for culturing due to its:

  • High individual risk (causes serious pulmonary and extrapulmonary disease).

  • Aerosol transmission potential (infectious via inhalation of droplet nuclei).

• BSL-3 protocols include:

  • Controlled access and medical surveillance for lab personnel.

  • Primary containment: Use of Class II biosafety cabinets for all procedures generating aerosols (e.g., centrifugation, culture manipulation).

  • Facility design: Negative airflow, HEPA-filtered exhaust, and two sets of self-closing doors.

Why Not the Others?

• a) BSL-1 → For non-pathogenic microbes (e.g., non-pathogenic E. coli).

• b) BSL-2 → For moderate-risk pathogens (e.g., Staphylococcus aureus), insufficient for M. tuberculosis due to aerosol transmission risk.

• d) BSL-4 → Reserved for lethal, exotic pathogens with no treatment (e.g., Ebola virus)

• Middlebrook 7H10 and 7H11 agar contain glycerol (2%), which can degrade over time into formaldehyde under light exposure or improper storage conditions. Formaldehyde is highly toxic to mycobacteria, inhibiting growth and viability 1316.

• Refrigeration (2–8°C) and protection from light are critical to prevent glycerol breakdown and formaldehyde accumulation, ensuring mycobacterial viability 411.

Why Other Options Are Incorrect:

• a) Blood agar and c) Chocolate agar: Used for fast-growing bacteria (not mycobacteria); no glycerol content, so formaldehyde production is irrelevant.

• d) Sabouraud dextrose agar: Designed for fungi; lacks glycerol and is not used for mycobacteria.

• PANTA is an antibiotic mixture used in liquid culture systems for mycobacteria (e.g., MGIT — Mycobacteria Growth Indicator Tube).

• It prevents the growth of contaminating bacteria and fungi while allowing mycobacteria to grow.

• PANTA stands for:

  • P: Polymyxin B → kills Gram-negative bacteria

  • A: Amphotericin B → antifungal

  • N: Nalidixic acid → kills Gram-negative bacteria

  • T: Trimethoprim → broad antibacterial

  • A: Azlocillin → kills Gram-negative bacteria

• These antimicrobials target likely contaminants but do not inhibit Mycobacterium tuberculosis.

Why not the others?

• (a) Enhance growth → Antibiotics do not enhance growth; they just prevent contamination.

• (c) Reduce incubation time → Liquid culture systems do that, but PANTA is not the reason.

• (d) Improve staining → Not related to staining; staining is a separate step.

• Oxalic acid is sometimes used in the digestion–decontamination process specifically for specimens heavily contaminated with Pseudomonas aeruginosa.

• This situation commonly occurs in cystic fibrosis (CF) patients, whose sputum often contains large numbers of P. aeruginosa.

• Oxalic acid effectively kills Pseudomonas without significantly affecting the viability of mycobacteria such as M. tuberculosis or M. abscessus.

Why not the others?

• (a) Blood → Blood cultures for mycobacteria are processed differently, no oxalic acid used.

• (c) Bone marrow → Sterile site, minimal contamination; no need for oxalic acid.

• (d) Urine → Not typically treated with oxalic acid; other decontamination methods are used if needed.

• The Xpert MTB/RIF assay is a rapid molecular test that detects:

  1. Mycobacterium tuberculosis complex DNA (diagnosing TB).

  2. Mutations in the rpoB gene, which confer rifampin resistance (a surrogate marker for potential multidrug-resistant TB [MDR-TB], as rifampin resistance often co-occurs with isoniazid resistance).

Why Not the Others?

• a) TB and HIV → HIV status requires separate testing (though Xpert may be used in HIV co-infected patients).

• c) TB and MDR-TB → While rifampin resistance suggests MDR-TB risk, the test does not directly detect isoniazid resistance (full MDR-TB confirmation requires additional testing).

• d) TB and leprosy → Xpert is specific for M. tuberculosis complex, not M. leprae.

• Mycobacterium leprae, the causative agent of leprosy (Hansen’s disease), is non-cultivable in artificial media (ruling out b) Middlebrook medium and d) MGIT).

• Ziehl-Neelsen (ZN) staining of nasal smears or skin biopsy specimens is the classic diagnostic method, where the bacilli appear as solid-staining, red rods (“cigar bundles”) against a blue background .

  • Fite-Faraco stain (a modified ZN method) is preferred for tissue biopsies due to better preservation of the bacilli’s lipid-rich cell wall .

Why Not the Others?

• b) Culture on Middlebrook medium → M. leprae cannot be cultured in vitro (requires armadillo or mouse footpad models).

• c) PCR from urine → Not standard; PCR is primarily used on skin biopsies or nerve tissue (e.g., RLEP gene target) but lacks widespread clinical validation .

• d) MGIT → Designed for M. tuberculosis; ineffective for M. leprae.