Ultrospec 2100

Kinetic measurements were performed at room temperature in HEPES buffer supplemented with zinc using a UV visible Ultrospec 2100 pro spectrophotometer (Amersham Biosciences, Buckinghamshire, UK).

Biofilms were grown on solid MSgg medium as indicated. The colonies were collected, resuspended in 1 mL PBS, and sonicated to remove the extracellular matrix (BRANSON digital sonifier, Model 250, Microtip, amplitude 20%, pulse 3x 5 sec). OD₆₀₀ had been measured (Ultrospec 2100, Amersham Biosciences. Cells (10⁸–10⁹) were taken for the assay. Cells were spun down, and pellets were resuspended in 1 mL of Z buffer (40 mM NaH₂PO₄, 60 mM Na₂HPO₄, 1 mM MgSO₄, 10 mM KCl, 38 mM β-mercaptoethanol) supplemented with 200 μg mL⁻¹ freshly made lysozyme. The samples were incubated for 15 min at 30 °C. Reactions were started by adding 200 μL of 4 mg mL⁻¹ ONPG (2-nitrophenyl β-D-galactopyranoside) and stopped by adding 500 μL of 1 M Na₂CO₃. The soluble fractions were transferred to cuvettes (VWR), and OD₄₂₀ values of the samples were recorded using a Pharmacia Ultraspectrometer 2000. The β-galactosidase-specific activity was calculated according to the equation [(OD₄₂₀/(time × OD₆₀₀)] × dilution factor × 1000. Time represents the time follwing the addition of ONPG. Assays were conducted in triplicates. For anaerobic growth and anoxic growth, biofilms were grown in anaerobic chamber or by the candle jar method^{50 (link)} in the presence of (KNO₃)^{27 (link),51 (link)}.

Yeast growth was monitored by measuring optical densities at 640 nm (OD_640nm) in an Ultrospec 2100 pro UV-visible (Amersham Biosciences®) spectrophotometer. Growth data were analyzed using the DMFit software available on the Combase website (http://www.combase.cc/index.php/en/). Growth data were fit to the model proposed by Baranyi and Roberts ([1994 (link)]) to obtain lag times and specific growth rates of each fermentation assay. Viability was determined throughout fermentation by counting colony forming units (CFU) on YPD solid medium, after 2–3 days of incubation at 28°C.

MTT stain was used to investigate cell viability and proliferation cultured as pellet in suspension culture (see 2.5.2). Cultures were performed in 15 ml falcon tubes at a density of 5 × 10⁴ cells/0.5 ml/tube up to 21 days. The experiment was repeated at least three times in triplicate on at least three different primary cell cultures. For the stain, tubes were centrifuged at 1,700g for 5 min, pellets were washed once with PBS, and 0.5 ml serum-free medium supplemented with 25 μl of MTT (Sigma) of 5 mg/ml stock solution was added to the tubes. Cells were incubated for 1 h in the dark. Then, pellets were washed twice with PBS. 1 ml of absolute ethanol was added to each tube; tubes were vortexed for 1 min to solubilize the purple formazan. The absorbance of the colorant was measured at 570/670 nm wavelength by using spectrophotometer Ultrospec 2100 (Amersham Biosciences).

Absorption spectra of coated- or uncoated-NPs were recorded using an UltroSpec 2100 spectrophotometer (Amersham Biosciences), a 1 cm path-length quartz cuvette, and buffer A or 5 mM sodium citrate buffer, pH 6.0, respectively, as blanks. The concentration of coated-NPs was calculated by interpolating the absorbance values at 530 nm on a calibration curve obtained using uncoated nanogold (stock solution: 3.3 × 10¹¹ NPs/ml, A₅₃₀ nm: 0.96 U/ml).
Transmission electron microscopy (TEM) analysis was performed using a TALOS L120C microscope (ThermoScientific) as described previously (Gasparri et al., 2019 (link)). Morphometric analysis of nanoparticle’s shape and diameter was performed using the ImageJ software, essentially as previously reported (Rice et al., 2013 (link)).

To determine culture density and live cell counts of cells grown in pellicles, cells were harvested from a floating biofilm assay (described above), and thoroughly vortexed. Untreated wild type cells were mildly sonicated at a BRANSON digital sonifier, Model 250, Microtip, with amplitude 20%, pulse 3 × 5 s (Thermo Scientific, Waltham, Massachusetts, USA). For culture density, OD₆₀₀ was measured with a Ultrospec 2100 spectrophotometer (Amersham Biosciences, Little Chalfont, England). To determine the number of live cell counts, cells were serial diluted in phosphate-buffered saline (PBS) (Biological Industries, Israel), plated on LB-plates, and colony forming units (CFU) were counted after incubation at 30°C over-night.

Ventral prostates were dissected under RNAse-free conditions. Thirty mg of the tissue was used for total RNA extraction. Subsequently, the tissue fragments were extracted using Illustra RNAspin Mini kits (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. RNA purity was analyzed by the ratio of absorbance at 260/280 nm (values higher than 1.8) and by electrophoresis on 1.2% denaturing agarose gel. The RNA concentration in each sample was determined in an Ultrospec 2100 pro spectrophotometer (Amersham Biosciences, Cambridge, England).