Carbon tab

A small film specimen was carefully deposited onto carbon tabs (Agar Scientific, Stansted, UK) and coated with carbon (Agar turbo carbon coater) to improve conductivity. Scanning electron microscope analysis (SEM) was performed on a FEI Quanta 3D 200 dual beam Focused Ion Beam Scanning Electron Microscope (FIB-SEM). The images were acquired using secondary electron imaging at an accelerating voltage of 5–15 kV.

To characterize particle size/shape, Scanning Electron Microscope EVO LS 15 (Zeiss, Rugby, UK) was used. The powders were scattered onto the surface of aluminum stubs (6 mm height/12.5 mm disc diameter) (Agar scientific, Stansted, UK) using carbon tabs (Agar scientific, UK), followed by a layer of gold coating (15 nm thickness). The samples were measured at four magnifications (30×, 100×, 500× and 1000×).

All P. aeruginosa strains were inoculated in 10 mL TSB and incubated overnight at 37 °C with constant shaking (200 rpm). After incubation, the optical density of the bacterial suspension was adjusted to 0.5 (OD_600 nm) and treated with ionic gold at the predetermined MIC concentration, for 2 h and 18 h. An untreated aliquot was used as a negative control. The cultures were centrifuged for 7 min at 1721 × g and washed with PBS. Aliquots of 25 μL of culture were placed on sterile stainless steel (316L) (1 cm × 1 cm) and dried for 1 h at room temperature. The samples were immersed in 4% glutaraldehyde (Agar Scientific, UK) at 4 ºC for 18 h. The samples were dehydrated by sequential submersion in an ethanol gradient of increasing concentrations (10%, 30%, 50%, 70%, 90% and absolute ethanol). The dehydrated samples were placed onto carbon tabs (Agar Scientific, UK). Samples were dried by desiccation over 24 h and were sputter coated with gold (Polaron, UK) for 30 s (parameters: power 5 mA, 30 s, 800 V, vacuum 0.09 mbar, argon gas) prior to imaging using a JEOL JSM 5600LV scanning electron microscope.