INICET Microbiology Previous Year Questions (2018–2026)

The correct answer is NaOH NALC (the NALC-NaOH method).

For mycobacterial (TB) sputum processing, the standard method is the NALC-NaOH method. N-acetyl-L-cysteine (NALC) is a mucolytic that digests and liquefies the mucus, and sodium hydroxide (NaOH) acts as the decontaminant, killing faster-growing commensal bacteria while the acid-fast mycobacteria survive. The specimen is then concentrated by centrifugation and the deposit used for smear and culture. So NaOH (with NALC) is correct.

The acids in the other options are not used for routine sputum digestion-decontamination. Strong acids (such as oxalic acid) are used only in special situations (for example to suppress Pseudomonas in cystic fibrosis samples), not as the standard mucolytic-decontamination step. NALC must be paired with NaOH, not with HNO3, HCl or H2SO4.

Incorrect Options:

• HNO3 NALC: nitric acid is not paired with NALC, and NaOH is the standard decontaminant.
• HCL NALC: HCl is used only to neutralise the NaOH after decontamination, not as the digesting/decontaminating agent.
• H2SO4 NALC: sulfuric acid is not used with NALC for routine sputum processing.

High-Yield Facts:

• Sputum for TB: NALC-NaOH method (digestion plus decontamination plus concentration).
• NALC = mucolytic (liquefies mucus), NaOH = decontaminant (kills commensals).
• After decontamination, concentrate by centrifugation, and HCl/buffer neutralises the NaOH.
• Mycobacteria survive because they are acid- and alkali-resistant.

Figure 1: Acid-fast bacilli in a Ziehl-Neelsen sputum smear - the smear made after NALC-NaOH digestion and concentration.

Figure 2: M. tuberculosis on Lowenstein-Jensen media - the concentrated deposit is cultured after decontamination.

The correct answer is NaOH and NALC.

Sputum is a thick, mucoid and heavily contaminated specimen, so before culturing Mycobacterium tuberculosis it must be liquefied (digested), freed of faster-growing flora (decontaminated) and concentrated. In the standard NALC-NaOH method, N-acetyl-L-cysteine (NALC) is the mucolytic agent that digests mucus and homogenizes the sample, while sodium hydroxide (NaOH, about 2 percent) is the decontaminant that kills contaminating bacteria as the acid-fast mycobacteria survive. Sodium citrate stabilizes NALC. The mixture is neutralized with phosphate buffer and centrifuged, which concentrates the bacilli into the sediment used for smear and culture. NaOH is kept dilute with a short contact time, because excess alkali also kills mycobacteria.

Incorrect Options:

• HNO3 and NALC: Nitric acid is not used for routine sputum decontamination. Strong mineral acids are reserved for other digestion settings, not the standard mycobacterial method.
• H2SO4 and NALC: Sulphuric acid is too harsh and would kill the mycobacteria, so it is not the paired digestant and decontaminant for sputum.
• HCl and NALC: HCl is at most used to neutralize residual NaOH afterwards, not as the decontaminating agent paired with NALC.

High-Yield Facts:

• NALC = mucolytic digestant, NaOH = decontaminant in the standard method.
• Standard NaOH is 2 percent (final about 1 percent) - higher concentrations kill mycobacteria.
• Bacilli are concentrated by centrifugation before smear and culture.
• Modified Petroff method uses 4 percent NaOH alone and is harsher.

Figure: Ziehl-Neelsen stained sputum smear - red, slender, curved acid-fast bacilli (M. tuberculosis), the smear prepared after NALC-NaOH digestion, decontamination and concentration.

The correct answer is pXO2 plasmid and poly-γ-D-glutamic acid capsule.

Bacillus anthracis is a highly virulent Gram-positive rod whose pathogenicity depends on two large plasmids: pXO1 and pXO2. The capsule is a critical virulence factor that confers resistance to phagocytosis. Unlike almost all other bacteria (which have polysaccharide capsules), B. anthracis possesses a unique polypeptide capsule composed of poly-γ-D-glutamic acid. This capsule is entirely encoded by the pXO2 plasmid.

The pXO1 plasmid encodes the tripartite anthrax toxins: Protective Antigen (PA), Edema Factor (EF), and Lethal Factor (LF). EF acts as a calmodulin-dependent adenylate cyclase (increasing cAMP), while LF is a zinc-metalloprotease that cleaves MAPKK (MEK). Both plasmids are required for full virulence; the Sterne vaccine strain is pXO1+ but pXO2-, meaning it is non-encapsulated and thus attenuated.

Incorrect Options:

• pXO1 plasmid: Incorrect for capsule; pXO1 encodes the toxins (PA, EF, LF) rather than the capsule synthesis machinery.
• Polysaccharide capsule: Incorrect biochemical nature; while typical for most bacteria (e.g., Pneumococcus), B. anthracis is the classic exception with its polypeptide layer.
• Poly-α-glutamic acid: Incorrect linkage; the capsule is specifically composed of gamma-linked (γ) D-glutamate polymers.

High-Yield Facts:

• B. anthracis capsule is the only medically important capsule made of polypeptide (poly-D-glutamate).
• Sterne strain (veterinary vaccine) lacks pXO2 and is therefore non-encapsulated.
• The McFadyean reaction (polychrome methylene blue) stains the capsule purple/red against blue bacilli.

Figure: India ink preparation (negative stain) highlights the thick antiphagocytic capsule as a transparent halo surrounding the dark-stained bacilli.