title: Analytical protocols for separation and electron microscopy of nanoparticles interacting with bacterial cells.
abstract: An important step toward understanding interactions between nanoparticles (NPs) and bacteria is the ability to directly observe NPs interacting with bacterial cells. NP-bacteria mixtures typical in nanomedicine, however, are not yet amendable for direct imaging in solution. Instead, evidence of NP-cell interactions must be preserved in derivative (usually dried) samples to be subsequently revealed in high-resolution images, for example, 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, for example, by SEM, for evidence of NP-bacteria interactions. These analytical protocols, validated by systematic control experiments and high-resolution SEM imaging, should be generally applicable for investigating NP-bacteria interactions.
authors.affiliation: †Center of Biological Engineering, University of Minho, Braga 4710-057, Portugal.
authors.forename: Cláudia
authors.initials: C
authors.lastname: Sousa
authors.affiliation: †Center of Biological Engineering, University of Minho, Braga 4710-057, Portugal.
authors.forename: Diana
authors.initials: D
authors.lastname: Sequeira
authors.affiliation: ‡International Iberian Nanotechnology Laboratory, Braga 4715-330, Portugal.
authors.forename: Yury V
authors.initials: YV
authors.lastname: Kolen'ko
authors.affiliation: ‡International Iberian Nanotechnology Laboratory, Braga 4715-330, Portugal.
authors.forename: Inês Mendes
authors.initials: IM
authors.lastname: Pinto
authors.affiliation: ‡International Iberian Nanotechnology Laboratory, Braga 4715-330, Portugal.
authors.forename: Dmitri Y
authors.initials: DY
authors.lastname: Petrovykh
mesh_terms: D002470:Cell Survival; D008854:Microscopy, Electron; D053758:Nanoparticles / Q000032:analysis*; D010316:Particle Size; D013211:Staphylococcus aureus / Q000166:cytology*; D013499:Surface Properties