Antimicrobial Activity Evaluation

Antimicrobial evaluation of herbal extracts follows the same disc diffusion and broth microdilution methodology used in synthetic antimicrobial screening. The disc diffusion (Kirby-Bauer) method inoculates a standardised microbial suspension (adjusted to 0.5 McFarland turbidity) onto Mueller-Hinton agar for bacteria or Sabouraud dextrose agar for fungi, applies extract-impregnated filter paper discs, and measures the resulting zone of inhibition following incubation, benchmarked against reference organisms such as Staphylococcus aureus, Escherichia coli, and Candida albicans and positive control antibiotic or antifungal discs. The minimum inhibitory concentration (MIC) is subsequently determined by broth microdilution, in which doubling dilutions of extract are tested against a standardised inoculum in a 96-well plate, with the addition of a resazurin viability indicator (which turns from blue to pink in the presence of viable, metabolically active organisms) commonly used to facilitate clear visual MIC determination. The minimum bactericidal concentration (MBC) is determined by sub-culturing wells at and above the MIC onto drug-free agar, with an MBC-to-MIC ratio of four or below conventionally taken as indicating bactericidal, rather than merely bacteriostatic, activity.