Fl 9496

Sample processing was performed using glass coverslips precoated with 1 mg/mL poly-l-lysine. Protozoa were fixed for 1 h in 2.5% glutaraldehyde diluted in 0.1 M cacodylate buffer pH 7.2. Cells were subsequently adhered to coverslips, postfixed for 1 h with 1% osmium tetroxide diluted in cacodylate buffer, and dehydrated in a graded alcohol series (50%, 70%, 90%, and two exchanges of 100% ethanol for 10 min each step). Samples were critical-point dried in a Leica EM CPD030 apparatus (Leica, Wetzlar, Germany). Specimens were sputtered with gold in a Balzers FL9496 unit (Postfach 1000 FL-9496 Balzers Liechtenstein) and observed in an EVO 40 VP SEM (Zeiss, Germany). In all assays performed, approximately 500 cells were observed.

In order to investigate the structural alterations of filamentous fungi following different treatments, SEM was used to check the cultured fungal colonies. For this experiment, the fungal fragments were processed according to Martinelli and Santos [22 ] and Mio et al. [23 (link)] using the Karnovsky solution (2.5% glutaraldehyde, 2.5% formaldehyde in 0.05 M sodium cacodylate buffer, and 0.001 M calcium chloride pH = 7.2) for 24 h. This was followed by further contact with a solution of 1% osmium tetroxide for 1 h in the dark and dehydration in increasing concentrations of acetone (25%–100%). Moreover, the fragment was fixed on the aluminum stubs with the aid of a double-sided carbon tape and was attached to the metallizer (Balzers FL-9496, Liechtenstein), with a gold steam bath for 180 s. A JEOL JSM 6390 LV field emission SEM (Tokyo, Japan) was used at an acceleration voltage of 15 KV. The analyses were performed at the Scanning Electron Microscopy Laboratory of the National Museum of the Federal University of Rio de Janeiro (Rio de Janeiro, Brazil).

The other half of the specimens was observed by SEM to surface morphological characterization. For this, the specimens were stored in a drying oven for 12 h, fixed in aluminum stubs with the aid of a double-sided carbon tape (Electron Microscopy Sciences, Washington, PA, USA), and coated with gold/palladium alloy on evaporator equipment (Balzers SCD 050 sputter coater, Balzers Union Aktiengesellschaft, and Fürstentum FL-9496; Balzers, Liechtenstein, Germany) by the 45 mA metallization process for 160 s. Specimens were analyzed on an SEM (Quanta 250; FEI, Hillsboro, OR, USA) at a voltage acceleration of 15 kV, 12 mm (work distance), and 20 nm spot-size lens aperture at 15,000× magnification.
In addition to SEM analysis, specimen chemical analyses were performed by EDS (EDS 6070; LEO Electron Microscopy/Oxford Microscopy, Cambridge, England). Thus, the qualitative microanalysis of the chemical elements might be present in each specimen and the chemical mapping of the dental surfaces.