An important step toward understanding interactions between nanoparticles (NPs) and bacteria is the ability to directly observe NPs interacting with bacterial cells. NPbacteria mixtures typical in nanomedicine, however, are not yet amendable for direct imaging in solution. Instead, evidence of NPcell interactions must be preserved in derivative (usually dried) samples to be subsequently revealed in high-resolution images, e.g., via scanning electron microscopy (SEM). Here, this concept is realized for a mixed suspension of model NPs and Staphylococcus aureus bacteria. First, protocols for analyzing the relative colloidal stabilities of NPs and bacteria are developed and validated based on systematic centrifugation and comparison of colony forming unit (CFU) counting and optical density (OD) measurements. Rate-dependence of centrifugation efficiency for each component suggests differential sedimentation at a specific predicted rate as an effective method for removing free NPs after co-incubation; the remaining fraction comprises bacteria with any associated NPs and can be examined, e.g., by SEM, for evidence of NPbacteria interactions. These analytical protocols, validated by systematic control experiments and high-resolution SEM imaging, should be generally applicable for investigating NPbacteria interactions.financial support from the following sources:
grant SFRH/BPD/47693/2008 from the Portuguese Foundation for Science and Technology (FCT); FCT Strategic Project PEst-OE/EQB/LA0023/2013; project “BioHealth Biotechnology
and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2−O Novo
Norte), QREN, FEDER; project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, ref. FCOMP-01-0124-FEDER- 027462
