Stains exploit differences in the bacterial cell wall: Gram staining separates thick-peptidoglycan from outer-membrane bacteria, and Ziehl–Neelsen detects the waxy mycolic acid wall of mycobacteria.
How it's asked: Order of Gram stain reagents, the step that differentiates, and acid concentrations used in modified ZN stains.
Why this is true
Crystal violet enters every cell and iodine fixes it as a large dye–iodine complex. The decolouriser is the deciding step: in Gram-positive bacteria it dehydrates the thick peptidoglycan mesh, shrinking the pores and trapping the complex, so they stay purple. In Gram-negative bacteria it dissolves the lipid-rich outer membrane, and the thin peptidoglycan cannot hold the complex, so the cells lose colour and take up the pink counterstain. Mycobacteria have mycolic acids that resist stain entry but, once stained with hot carbol fuchsin, also resist decolourisation by acid — they are 'acid-fast'.
Key points
Gram stain, step by step
- 1Crystal violetEverything stains purple
Common traps
- Over-decolourising makes Gram-positives look Gram-negative; old cultures can also stain variably.
- Mycobacteria are technically Gram-positive but stain poorly — use ZN or auramine.
Clinical case
A skin smear from a patient with thickened ulnar nerves shows red bacilli in clumps after ZN staining with 5% sulphuric acid.
High-yield
Gram: CV → iodine (mordant) → decolouriser (critical step) → safranin. ZN acid: 20% M. tb, 5% M. leprae, 1% Nocardia. Albert's = diphtheria granules.
Quick check
Q1.The mordant in Gram staining is:
Q2.Concentration of sulphuric acid used to decolourise M. leprae in ZN staining: