Pelco grids

Early log phase cultures of S. aureus and S. enterica were subjected to different concentration of peptides and incubated at 37°C. The bacterial pellets after centrifugation (5000 g for 10 min) were washed with 0.1M cacodylate buffer (pH 7.2) and were fixed in 2.5% gluteraldehyde at 4°C for 2 h. For scanning electron microscopy, bacteria were dehydrated using gradient series of ethanol wash (30, 50, 70, 90 and absolute). The samples were air-dried overnight, gold coated (Eiko IB-3 ion coater), and viewed in a Zeiss EVO 18 Special Edition SEM. For transmission electron microscopy, fixed samples were adhered to hexagonal PELCO® Grids (Ted Pella, Inc. USA), washed once with Mili-Q water and stained with uranyl acetate (2%) buffer. Samples were viewed in a JEM 1011 (Jeol, Japan) microscope.

All nanoparticles were characterized by JEM CX-100 transmission electron microscope (Jeol, Tokyo, Japan) at an accelerating voltage of 80 kV. Nanoparticles were dropped onto a grid (300 mesh, PELCO Grids, Ted Pella, Redding, CA, USA) coated with polyvinyl formal support film. The obtained images of nanoparticles were digitalized and processed with Image Tool software (University of Texas Health Science Center in San Antonio, TX, USA). To estimate the size distribution using the TEM data, single-peaked Gauss approximation was used. The fitting was calculated using OriginPro 9.0 software (OriginLab, Northampton, MA, USA). Meanwhile, using the image of nanoparticles from TEM micrographs, we calculated the ellipticity coefficient by the ratio of major axis to its minor axis. For each type of nanoparticle, mean values and standard deviations of coefficient of ellipticity were found.

The size and morphology of the nanocarriers were determined by transmission electron microscopy (TEM; FEI T12 Tecnai™ T12 Spirit, Hillsboro, OR) at OHSU's Multiscale Microscopy Core. The nanocarrier dispersion was negatively stained with 1% uranyl acetate for TEM measurements. One 10 μL drop of an aqueous dispersion specimen was deposited on a carbon-coated TEM copper grid (PELCO® Grids, Ted Pella Inc., Redding, CA) with 300 mesh and allowed to dry in air for 2 min. The surface of the carbon film had previously been glow-discharged by exposure under plasma to render it hydrophilic.
Immediately before TEM sample preparation, the nanocarriers were vortexed for 60 s, vigorously pipetted, ultrasonicated for 10 min in the presence of ice, and vortexed again for an additional 10 s, which helped break down nanocarrier aggregates.