Q150t es turbo pumped sputter coater

SEM micrographs were taken on calla lily and cabbage leaves following disinfection in 0.7% sodium hypochlorite for 15 min and a double wash with sterile DDW. Bacterial cultures (PC16 and WPP14) that had been grown overnight were centrifuged and re-suspended in sterile DDW at 10⁸ cfu/mL and 10 µL of that suspension were placed on the leaf surface and incubated at 28 °C for 3 h inside an airtight box. Leaf samples (4 × 4 mm), from bacterial inoculation site, were gently rinsed with DDW, then fixed in 70% ethanol overnight, followed by dehydration with 90%, 95%, 100% ethanol for 1 h each and a second dehydration with 100% ethanol. Then, the samples were dried on a K850 critical-point dryer and coated with gold-palladium alloy in a Q150T ES turbo-pumped sputter coater, following the manufacturer’s instructions (Quorum Technologies Ltd., East Sussex, UK). Finally, samples were observed under SEM, JSM-7800F (JEOL Inc., Peabody, MA, USA). Experiment was performed with five biological repeats and 10 replicates for each strain-host combination. The micrographs are representative photos of the experiment. In addition, 5 leaves were inoculated as above, and 3 h post inoculation were washed gently in DDW, and sections (4 × 4 mm) were excised and grind in 200 µL sterile DDW, serial dilutions were made and plated onto LB agar for CFU assessment.

For each group, two teeth were prepared to be analyzed by SEM (Scanning Electron Microscopy). The dentin surface was exposed, and the adhesives and resin build-ups were applied according to the same procedure described in Section 2.2. The teeth were then stored in distilled water at 37 °C for 24 h. After this period, samples were immersed in a glutaraldehyde and sodium cacodylate fixation solution according to Perdigão et al. [24 (link)]. The specimens were sectioned with a hard tissue microtome (Accuton 50, Struers A/S, Ballerup, Denmark) in a perpendicular plan to the adhesive interface, and the un-hybridized dentin was removed after demineralization and deproteinization procedures [25 (link)]. They were then dehydrated using increasing concentrations of ethanol (70%, 95% and 100%) and subsequently dried by immersion in methyldisilazane (HMDS) [24 (link)].
Finally, samples were subjected to a metallization process, where each one was placed on a self-adhesive double-sided carbon tape and then coated with a thin film of approximately 8 nm of Au/Pd on a Q150T ES Turbo-Pumped Sputter coater (Quorum Technologies, Q150T Turbomolecular-pumped coating system, UK). The hybrid layer was then observed at SEM (FEG-SEM JEOL, model JSM7001F, Tokyo, Japan) using 500× and 2000× magnifications.

For each experimental group, two additional teeth per experimental group stored and prepared under the same conditions as described in Section 2.2, were selected for visualization of the bonding interface using scanning-electron microscopy (SE, FEG-SEM JEOL, model JSM7001F, Tokyo, Japan).
After 24 h in an incubator, the specimens were fixed with glutaraldehyde (2.5%) and 0.1 M of sodium cacodylate, at 4 °C, for 24 h [19 (link)]. Subsequently, the specimens were sectioned exposing the adhesive interface with a hard tissue microtome (Accuton 50, Struers A/S, Ballerup, Denmark) and a protocol of demineralization and deproteinization was performed in order to expose the resin tags and remove sectioning debris [20 (link)]. Ethanol in ascending concentrations was then used (70%—20 min; 95%—20 min and 100%—30 min, 3×), followed by hexamethyldisilazane (HMDS) in two immersion periods of 10 min each. After 24 h of air-drying, samples were then fixed with a carbon tape and sputter coated with a thin film of gold/palladium on a Q150T ES Turbo-Pumped Sputter coater (Quorum Technologies, Q150T Turbomolecular-pumped coating system, Lewes, UK) with a 15.0 kV electron acceleration beam. The adhesive interface was analyzed using low and high magnifications (500× and 2000×) to visualize the ultrastructure of the hybrid layer.