Hummer sputtering system

Spot assays were performed as described above by spotting 5 µL fresh full-length Gp047 in PBS at 0.91 mg/mL. Following overnight growth of lawns, agar squares were excised using a sterile scalpel from either inside, outside or at the interface of the clearance zone. Slabs were then trimmed to leave the top layer (approximately 2 mm) intact and incubated in scanning electron microscopy (SEM) fixative (2.5% glutaraldehyde; 2% paraformaldehyde in 0.1 M phosphate buffer) overnight at 4 °C. Slabs were then prepared for microscopy using methods described by Bozzola and Russell [29 ]. Briefly, slabs were washed three times in 0.1 M phosphate buffer for 10 min each and then dehydrated by incubating 15 min each in a series of alternating ethanol and hexamethyldisialazane (HMDS) washes. The washes were done as follows: 50% ethanol, 70% ethanol, 90% ethanol, 100% ethanol, ethanol:HMDS 75:25, ethanol:HMDS 50:50, ethanol:HMDS 25:75 and 100% HMDS. Slabs were incubated in just enough HMDS to cover the slab surface, which was left to evaporate overnight. Once fully dried, slabs were mounted onto SEM stubs, sputter-coated with gold using the Hummer sputtering system (Anatech Ltd., Battle Creek, MI, USA) and imaged using the Philips/FEI (XL30) scanning electron microscope (Philips/FEI, Hillsboro, OR, USA) with an electron beam energy of 20 kV.

Surface features of the extruded tubes comprising PCL homopolymers and binary blends were visualized by scanning electron microscopy (Quanta 200; FEI, Hillsboro, OR, USA) at an accelerating voltage of 20 kV and a spot size of 2.5 under high vacuum at various magnifications. Samples were prepared for imaging by slicing the extruded tubes and flattening them onto a carbon adhesive substrate mounted to an aluminum sample holder sputter coated with Au/Pt (Hummer Sputtering System, Anatech Ltd., Union City, CA, USA) under argon for 120 s. The surface morphology of the PCL extruded tubes was also characterized using an Olympus SZ61 stereomicroscope (zoom range 0.67× to 4.5×). The microscope is equipped with an Olympus SC30 camera that allowed sample photography. Similarly, the extruded tubes were sliced open and flattened to adhere to glass slides.

Emitter tips were gold coated using a Hummer sputtering system (Anatech USA, Union City, CA, USA). SEM images were obtained using a FEI-MLA (Hillsboro, OR, USA) Quanta 650 Field Emission Gun-Environmental SEM.

Following a clearance assay with CTB on C. jejuni HS:19 as described above, agar squares were excised from inside, outside or at the interface of the clearance zones and prepared using the following established protocol^{58 (link)}. The squares were excised using a scalpel and trimmed to leave only the top layer of agar (about 2 mm) and then incubated in scanning electron microscopy (SEM) fixative (2.5% glutaraldehyde; 2% paraformaldehyde in 0.1 M phosphate buffer) overnight at 4 °C. Excised squares were then washed in 0.1 M phosphate buffer three times for 10 min and dehydrated using a series of 15 min washes: 50% ethanol, 70% ethanol, 90% ethanol, 100% ethanol, ethanol: hexamethyldisilazane (HMDS) 75:25, ethanol:HMDS 50:50, ethanol:HMDS 25:75 and 100% HMDS. After leaving HMDS to evaporate overnight, the excised squares were mounted onto SEM stubs and sputter-coated with the Hummer sputtering system (Anatech Ltd.). The squares were then imaged using the Phillips/FEI (XL30) scanning electron microscope (Philips/FEI) with an electron beam energy of 20 kV.