Standardizing and optimizing disk inhibition zone testing against E. coli for reproducible evaluation of antibacterial polymeric membranes

The disk inhibition zone test is a widely used and visually interpretable method for evaluating the antibacterial properties of membranes. However, the lack of a consistent, standardized methodology for disk inhibition zone testing limits reliable assessment of antibacterial performance in membranes. Herein, we introduce a systematic and reproducible framework for investigating the key experimental parameters, including bacterial concentration (10 5 –10 9 CFU/mL), nutrient media (i.e., tryptic soy broth (TSB)) concentration (15–60 g/L), incubation time (6–10 h), and various bacterial spreading techniques such as overlayer (OL) glass spreader (GS), and cotton swabs (CS) methods. Quantitative image analysis in ImageJ, combined with comprehensive statistical evaluation, revealed that bacterial concentration and TSB levels were the most influential factors in determining inhibition zone diameter. In contrast, the choice of spreading technique significantly affected the uniformity and integrity of the inhibition zone. Results showed that utilizing a bacterial concentration of 10 7 CFU/mL, TSB concentration of ≥ 30 g/L, incubation times of 8- 10 h, and the Overlayer (OL) method, although less commonly used in literature, led to the most consistent inhibition zones, as these conditions promote stable bacterial growth kinetics and uniform lawn formation. This framework provides a reliable approach to standardize antibacterial membrane testing and improve cross-study comparability and validation.