Aims: Engineered metal nanoparticles are increasingly used in consumer products, in part as additives that exhibit advantageous antimicrobial properties. Conventional nanoparticle susceptibility testing is based largely on determination of nontemporal growth profiles such as measurements of inhibition zones in common agar diffusion tests, counting of colony-forming units, or endpoint or regular-interval growth determination via optical density measurements. For better evaluation of the dynamic effects from exposure to nanoparticles, a cultivation-based assay was established in a 96-well format and adapted for time-resolved testing of the effects of nanoparticles on microorganisms. Methods and Results: The modified assay allowed simultaneous cultivation and on-line analysis of microbial growth inhibition. The automated highthroughput assay combined continuous monitoring of microbial growth with the analysis of many replicates and was applied to Cupriavidus necator H16 test organisms to study the antimicrobial effects of spherical silver [Ag(0)] nanoparticles (primary particle size distribution D90 < 15 nm). Ag(0) concentrations above 80 lg ml À1 resulted in complete and irreversible inhibition of microbial growth, whereas extended lag phases and partial growth inhibition were observed at Ag(0) concentrations between 20 and 80 lg ml À1 .Addition of Ag(0) nanoparticles at different growth stages led to either complete inhibition (addition of 40 lg ml À1 Ag(0) from 0 h to 6 h) or resulted in full recovery (40 lg ml À1 Ag(0) addition ! 9 h). Conclusions: Contrary to the expected results, our data indicate growth stimulation of C. necator at certain Ag(0) nanoparticle concentrations, as well as varying susceptibility to nanoparticles at different growth stages. Significance and Impact of the Study: These results underscore the need for time-resolved analyses of microbial growth inhibition by Ag(0) nanoparticles. Due to the versatility of the technique, the assay will likely complement existing microbiological methods for cultivation and diagnostics of microbes, in addition to tests of other antimicrobial nanoparticles.

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