Vibra cell 400w

The liposomal formulations were next tested for their inhibitory effects on biofilm viability, as previously described (Graziano et al., 2015 (link)) (39). Cellulose acetate membranes (25 mm diameter, 0.2 µM pores) (Sartorius Stedim GmbH, Guxhagen, Hessen, Germany) were used as substrates for S. aureus biofilm formation. The membranes were placed in 6-well plates containing BHI medium supplemented with 1% glucose and bacterial suspension (approximately 1 × 10⁶ CFU/ml in each well). The plates were incubated at 37°C for 24 h. Then the membranes were transferred to new plates containing fresh BHI plus 1% glucose, and biofilms were treated with the formulations at 1 × MIC, 10 × MIC, and 50 × MIC for 24 h. Treated biofilm-coated membranes were gently washed (three times) through immersion into 5 ml of 0.9% NaCl. Then, the membranes were transferred to other tubes containing freshly 5 ml of 0.9% NaCl and then sonicated with six pulses of 9.9 s, 5 s time-interval, 5% amplitude (VibraCell 400W, Sonics & Materials Inc., Newtown, CT, USA) and vortexed at 3,800 rpm for 30 s. Ten-microliter aliquots were collected from each tube, serially diluted, and plated for CFUs onto TSA. The plates were incubated at 37°C for 24 h.

To assess the effect of simvastatin on the viability of mature biofilms, 24-h biofilms were exposed to the antimicrobial drugs. Biofilms of S. aureus ATCC 29213 were formed in cellulose acetate membranes filters (diameter: 25 mm; pore size: 0.2 μm; Sartorius AG, Germany) placed at the bottom of 6-well plates.
After biofilm formation, membranes were transferred to a new plate containing culture media and the antimicrobial substances at 4xMIC. The plates were then incubated for 24 h at 35°C. Membranes were removed from the plates and washed three times with 5 mL of 0.9% NaCl for 5 s each. Then, membranes were transferred to polystyrene tubes containing 5 mL of 0.9% NaCl and sonicated (Vibra Cell 400W, Sonics & Materials Inc., Newtown, CT, USA) in the 5% range and 20 W for 30 seconds for dispersion of the biofilm. Samples of 10 μL were collected, plated on TSA and kept at 35°C for 24 h. After this period, viable counts were performed.

Primary particle size, and the morphological and structural characteristics of ZnO NPs were investigated by transmission electron microscopy (TEM). Samples were prepared by drop-casting dispersions of the NPs onto carbon-coated Cu grids after a 3 min sonication with an ultrasonic (VibraCell 400 W, Sonics & Materials Inc., Newtown, CT, USA), applying a microtip probe under intensity settings 4 [36 (link)]. Finally, TEM images were obtained with a Jeol JEM 1010 microscope (Jeol, Tokyo, Japan) [37 (link)].
Dynamic light scattering (DLS) analysis was used to define the size-distribution profile of ZnO NPs (5 mg L⁻¹, 10 mg L⁻¹ and 50 mg L⁻¹). Zeta (ζ) potential measurements were conducted to assess the surface charge of the particles as described previously [5 (link)]. All measurements were performed in Milli-Q water after brief sonication at 25 °C [5 (link)]. Results are presented as means (±SD) of three measurements.