Su5000

For scanning electron microscopy (SEM), deposited glass slides (Lab-Tek and Shell-vial 12 mm) were fixed with glutaraldehyde 2.5% in 0.1 M of sodium cacodylate buffer for 60 min. Slides were rinsed with 0.1 M sodium cacodylate buffer and distilled water for 1 minute each. Slides were dehydrated with increasing ethanol solutions (30%, 50%, 70%, 90%) for 2 minutes and with 100% ethanol for 5 minutes. Slides were incubated with 100% ethanol/100% hexamethyldisilazane (HDMS) in a 1:2 ratio for 5 minutes. Slides were incubated with 100% of HDMS for 5 minutes and air-dried for 30 min. We checked that no particles had been lost after this virus preparation (dehydration and drying) for SEM (not shown). Finally, the slides were platinum sputter-coated for 20 s at 10 mA (Hitachi MC1000). The observation was made using a SU5000 (Hitachi High-Technologies, Tokyo, Japan) SEM with an SE detector in high-vacuum mode at 1 kV acceleration voltage, observation mode (spot size 30). The working distance ranged between 1 mm and 5 mm. For quantification, automatic 6 × 6 mosaic tiled images at ×5k magnification were acquired at a random position with auto contrast/brightness using the microscope zig-zag function after manual adjustment of the focus and no further auto-focus. Pixel size was 3.96 nm. Images were 1280 × 960 pixels corresponding to 5078 × 3808 nm fields of view.

The samples were characterized using different techniques to determine their structure and morphology. Fracture sections of the specimens before and after they underwent hygrothermal aging were observed using scanning electron microscopy (SEM, SU5000 type, Hitachi High-Technologies, Kyoto, Japan). For the SEM, the specimens were place in liquid nitrogen, taken out, and quenched quickly. Gold (SBC-12, KYKY, Beijing, China) was sprayed on the fracture, and the filler distribution in the specimens was observed.
For a dynamic mechanical analysis (DMA) using a DMA-8000 (Perkin-Elmer Corporation, Waltham, MA, USA), the specimens were cut into rectangles of 20 mm × 5 mm × 2 mm. The DMA experiments were conducted with the specimens before and after they underwent hygrothermal aging. This test was performed through a single cantilever beam experiment at a vibration frequency of 1 Hz and an amplitude of 0.05 mm. The heating rate was 5 °C/min, and the experimental temperature was −50~150 °C. Plots of the storage modulus and $\tan \delta$ vs. temperature were recorded, and the $T_g$ was obtained from the $\tan \delta$ peak.

To evaluate the effects of CARB and Cu²⁺ on bacterial morphology, scanning electron microscopy (SEM) was performed. In this study, the MIC was 2 μg/mL of CARB and 1,600 μg/mL of Cu²⁺, and the inhibitory effect of biofilm was observed at sub-MIC that could not kill the bacteria. Thus, the concentrations used in this study were 1/4 MIC and 1/2 MIC. The prepared 2.5 mL samples (1/4, 1/2 MIC CARB, and Cu²⁺) were placed in 15 mL conical tubes containing 2.5 mL TSB of bacterial solution (~6 Log CFU/mL). Finally, they were mixed well using a vortex mixer and incubated at 37°C for 16 h. Afterwards bacterial suspension was centrifuged for 10 min at 3000 g. The resultant pellets were mixed with 1 mL glutaraldehyde (2.5%) for overnight at 4°C, after which the bacterial were washed several times with PBS, sequentially dehydrated with 30, 50, 70, 90, and 100% (v/v) ethanol, and 100% (v/v) ethanol was used twice for 10 min. The specimens were coated with gold for observation by SEM (SU5000, Hitachi High-Tech, Japan).

This study observed the microscale interactions between XG and kaolinite under dry and humid conditions, the relative humidity being 60% and 100%, respectively, using SEM (SU-5000, Hitachi High Technologies) and ESEM (Model Quattro ESEM, Thermo Fisher Scientific Inc., Waltham, USA). Untreated and treated kaolinites—that is, m_b/m_s = 0, 1.0%—at w = 35% were air-dried for 24 h at 20 °C and attached to a 25-mm diameter SEM mount using carbon conductive tabs (PELCO Tabs; Ted Pella, Inc.). Before SEM observations, the specimens were osmium (OsO₄)-coated for 10 s under a vacuum using a plasma coater (OPC-60A).
The ESEM, which controls the water vapor pressure (10–4000 Pa) and the relative humidity in its specimen chamber^{96 (link)}, was used to assess the variation of XG-treated kaolinite (m_b/m_s = 1%) using a change in the relative humidity. The samples (w = 35%) were initially attached to an ESEM mount, and the specimen surfaces were exposed to the electron beams thereafter. During the observations, the relative humidity fluctuated between 0 and 100%.

Bacteria were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for at least one hour. After fixation, bacteria were rinsed for one minute with 0.1 M sodium cacodylate. Bacteria were gradually dehydrated with increasing ethanol concentrations: 30%, 50%, 70%, 90%, and 100% (one minute each). Bacteria were incubated for one minute in ethanol/hexamethyldisilazane (HMDS, Sigma Aldrich, USA) with a 1:2 ratio and finally incubated in pure HMDS. Between all previous steps, cells were gently stirred and centrifuged at 5000 rpm. Finally, 100 µL of each bacteria solution was centrifuged on a cytospin glass slide at 800 rpm for eight minutes. After deposition, bacteria were air-dried for five minutes and slides were platinum sputter-coated for 20 s at 10 mA (Hitachi MC1000). Observations were made using a SU5000 (Hitachi High-Technologies, Tokyo, Japan) SEM with Secondary-Electrons (SE) detector in high-vacuum mode at 1 kV acceleration voltage, observation mode (spot size 30).