Metal oxide nanoparticles (MO-NPs) are known to effectively inhibit the growth of a wide range of
Gram-positive and Gram-negative bacteria. They have emerged as promising candidates to challenge the rising
global issue of antimicrobial resistance. However, a comprehensive understanding of their mechanism of action
and identifying the most promising NP materials for future clinical translation remain a major challenge due to
variations in NP preparation and testing methods. With various types of MO-NPs being rapidly developed, a
robust, standardized, in vitro assessment protocol for evaluating the antibacterial potency and efficiency of these
NPs is needed. Calculating the number of NPs that actively interact with each bacterial cell is critical for assessing
the dose response for toxicity. Here we discuss methods to evaluate MO-NPs antibacterial efficiency with
focus on issues related to NPs in these assays. We also highlight sources of experimental variability including NP
preparation, initial bacterial concentration, bacterial strains tested, culture microenvironment, and reported dose.
Keywords: Metal oxides, bacteria, nanoparticles, assembly/agglomeration, antibiotic resistance, dose response.
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