S 3400

Small strips of 2 × 5 mm were cut from the center of yogurt stored for 21 d and fixed in 2.5% glutaraldehyde solution (pH 6.8) for 6 h. After fixation, the samples were rinsed three times with phosphate buffer (pH 6.8) and then dehydrated with 50%, 70% and 90% ethanol, respectively, and twice with 100% ethanol for 10 min each. The samples were frozen at −20 °C for 30 min and dried in a freeze dryer (ES-2030, Hitachi, Japan), and the dried samples were fixed on a sample table with tape and coated with an ion sputter coater. The samples were observed and photographed using a scanning electron microscope (SEM) S-3400 (S-3400, Hitachi, Japan).

The biofilm formation ability of A. baumannii strains was visualized by scanning electron microscope (SEM) (Hitachi-S3400, Tokyo, Japan). Biofilm was formed on the minimum biofilm eliminating concentration device (MBEC™ P&G Physiology & Genetics Innovotech, Alberta, Canada). Briefly, A. baumannii suspensions (200 μL) were inoculated into each well and then incubated overnight at 37 °C. Biofilms that formed were then washed twice with PBS to remove any unattached and floating cells and were fixed with 2.5% glutaraldehyde in 0.1 M cacodylic acid (pH 7.2) at 4 °C for 24 h and post fixed with 0.1 M cacodylic acid for approximately 10 min. After incubation, the plates were washed twice with distilled water for 15 min, followed by gradual dehydration with ethanol, and air dry for a minimum of 24 h. The fixed biofilms were then coated with a layer of gold–palladium (7 nm thick) and examined with SEM (Hitachi-S3400) [36 ].

The textural and morphological properties of the polystyrene microsphere templates were observed on a scanning electron microscope (Hitachi High Technologies America Inc. S-3400 Los Angeles, CA, USA) operated at 10 kV. Samples deposited on the sample holder were coated with a conductive gold before morphological examination. Successful incorporation and removal of the microsphere template on the monolith were also confirmed by observing the monoliths using SEM. The average size of the polystyrene microsphere particles at different concentrations was determined using Dynamic Light Scattering (Nanoplus Micromeritics Instrument Corp, Tewkesbury, UK). The average pore size and the pore distribution of the monoliths obtained using different ratios were evaluated using Image J, 1.52 version software on the pre-captured image from SEM observation (Chan et al., [5 (link)]). The original SEM image was uploaded and binarized by altering the threshold. Particle size measurements were carried on based on the created particle outline. Fourier transform infrared spectroscopy (Agilents Technology Cary 630, Santa Clara, CA, USA) was used to characterize the functional groups in the monoliths after the incorporation and removal of the templates. The changes were observed by comparing the tested monoliths with a control monolith synthesized without the presence of a template.