Jsm 6390lv

The morphology of both the cell-free and cell laden scaffolds was investigated by means of scanning electron microscopy (JEOL JSM-6390 LV) at days 2 and 10. The cell-laden scaffolds were rinsed twice with PBS and fixed using a PFA 4% solution. The scaffolds were then dehydrated by employing increasing ethanol concentrations, from 30 to 100% pure ethanol. For the cell-free scaffolds, the same procedure was followed but without fixation. At the last stage, the scaffolds were dried in a critical point drier (Baltec CPD 030), sputter-coated with an 80 nm thick layer of gold (Baltec SCD 050) and observed under a scanning electron microscope at an accelerating voltage of 20 kV (JEOL JSM-6390 LV).

Cell adhesion and morphology on the ceramic samples was evaluated by means of scanning electron microscopy (SEM, JEOL JSM-6390 LV, Tokyo, Japan). Pre-osteoblastic cells were seeded on each of the five ceramic samples at a density of 1 × 10⁵ cells/sample in 24-well plates and cultured for 2 and 14 days in a humidified incubator at 37 °C. All samples were fixed by addition of 4% paraformaldehyde (PFA), dehydrated with increasing concentration of 20, 50, 70, 90 and 100% ethanol that was exchanged with liquid carbon dioxide by critical point drying (Baltec CPD 030, Baltec, Los Angeles, CA, USA). The dry samples were sputter-coated with gold (Baltec SCD 050) to increase their conductivity, and were then placed under the scanning electron microscope (JEOL JSM-6390 LV, Tokyo, Japan) for observation at a voltage of 20 kV.

For observation under SEM, we adjusted the bacterial suspensions to 10⁷ cfu mL^–1 in luria broth and added suspensions of the two variations of AgNPs to each bacterial sample to a final concentration of 75 μg/mL. After 5, 10, or 24 h of incubation, a 100 μL aliquot was placed on filter membranes with a 0.2 μm pore size. After rinsing with 0.1 M sodium cacodylate (SCB) buffer at pH 7.4, the samples were fixed with 2% paraformaldehyde and glutaraldehyde solution for 30 min. The samples were then washed twice with sodium cacodylate buffer and gradually dehydrated with increasing ethanol concentrations of 30%, 50%, 70%, 90%, and 100% (v/v) ethanol in water for 10 min, respectively. The samples were subsequently dried in a critical point drier (Baltec CPD 030), sputter-coated with a 10 nm thick layer of gold (Baltec SCD 050) and observed under a scanning electron microscope (JEOL JSM-6390 LV) (JEOL, Peabody, MA, USA) at an accelerating voltage of 25 kV.

Kidneys were collected, cut into small tissue blocks (1 mm³), and fixed in 2.5% glutaraldehyde at 4 °C. For transmission electron microscope, the fragments were washed in 0.1 M sodium cacodylate buffer, after, postfixaded with 2% osmium tetroxide, tissues were dehydrated in increasing series of acetone (30, 50, 70, 90 and 100%), 15 min each step, and embedded in epoxy resin for three days at 60 °C. Ultrathin sections were contrasted with uranyl acetate and lead citrate. Sections were examined with a JEM1011 electron microscopy (JEOL, Akishima, Tokyo, Japan).
For scanning electron microscopy, the fragments were washed in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide diluted in 0.1 M sodium cacodylate buffer. After further washing, the material was dehydrated in an increasing series of ethanol (30, 50, 70, 90% and 2× absolute), 30 min each step. The material was taken to the critical point device for the replacement of ethanol by CO₂, later fixed in stubs with carbon tape and metallized with gold. After metallization, the material was analyzed using a scanning electron microscope JEOL-JSM-6390-LV (JEOL, Akishima, Tokyo, Japan).

Two membranes per group of each experimental period were analyzed by scanning
electron microscopy (SEM) (JEOL JSM-6390 LV; JEOL Ltd., Tokyo, Japan) to assess
the biofilm architecture formed in the membrane surface ²⁸. The membranes were rinsed with 0.9% sterile saline baths (3 × 1 mL) and
fixed in 2.5% glutaraldehyde buffered with 0.2 M cacodylate at 4 °C for 12 h.
After being washed with cacodylate buffer for 1 h and dehydrated with increasing
grades of ethanol (25%, 50%, 75%, and 95% for 20 min for each concentration, and
100% for 1 h), they were dried via critical point drying method (EM CPD 030 /
LEICA). The membranes were mounted on metallic stubs with the superior surface
facing upwards and sputter-coated with a gold layer (300 Å). Two representative
areas of each membrane were selected to assess the morphology and organization
of the biofilm over the membrane fibers. The images were photographed from 300 ×
up to 3.000 × magnification, with the SEM operating the 10 kW.

Scanning electron microscopy (SEM) was carried out using a JEOL JSM-6390LV (JEOL USA, Inc., Peabody, MA, USA) scanning microscope (0.5–30 kV) with a high resolution of 3 nm equipped with an energy-dispersive X-ray (EDX) INCAPentaFETx3 (Oxford Instruments, Abingdon, UK) microanalytical system. All the studied surfaces were coated with carbon black to avoid charging under the electron beam. The samples were probed with a beam of electrons focused into a spot on the sample surface and Smile Shot^TM software was used to capture the microphotos.