Regarding disc diffusion (Kirby-Bauer) antibiotic susceptibility testing, which statement about inoculum preparation is most critical for accurate results?
- A The inoculum must be derived from a single colony, not a broth culture, to prevent mixed inhibition zones
- B The inoculum must be adjusted to 0.5 McFarland turbidity standard (approximately 1–2 × 10⁸ CFU/mL) before plating on Mueller-Hinton agar ✓
- C An inoculum density of 1.0 McFarland is required for all organisms including Streptococcus and Haemophilus
- D The inoculum must be heat-killed before application to prevent biosafety risk from disc diffusion plates
Explanation
The CLSI and EUCAST standard for Kirby-Bauer disc diffusion requires inoculum turbidity adjusted to match the 0.5 McFarland standard (1–2 × 10⁸ CFU/mL for most organisms). Insufficient inoculum produces falsely large inhibition zones (falsely susceptible), while excess inoculum produces falsely small zones (falsely resistant). Mueller-Hinton agar is the standard medium (pH 7.2–7.4, calcium and magnesium content standardized). Different inoculum densities (e.g., 0.5 McFarland for Enterobacterales/S. aureus) are used per organism group. Using a single colony is part of obtaining a pure culture but does not replace McFarland standardization. Plates are incubated with live organisms.
Reference: Ananthanarayan & Paniker's Textbook of Microbiology, 11th ed.
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