Genesys 150 spectrophotometer

The barrier properties of films against ultraviolet and visible light were assessed according to the methodology followed by Dick et al. [18 (link)]. For this purpose, films were cut into rectangular pieces of 1 cm width and placed inside a glass spectrophotometer cell. The light transmission of the samples was tested using a UV-Visible Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA), from 280 to 800 nm, using an empty test cell as a blank. The transparency of the films was calculated according to the Equation (2):
where A₆₀₀ is the absorbance of the film sample at 600 nm and x is the film thickness (mm).
Film thickness was measured with a micrometer (Mitutoyo, Kanagawa, Japan), with a precision of ±1 µm. The thickness was measured in seven different areas, one of them in the centre of the film and the other six around the film perimeter.

For the determination, 150 μL of sample was mixed with 2850 μL of the previously prepared 2,3,5-triphenyltetrazolium chloride (TPTZ) reagent, which was left for 30 min at 37 °C. The absorbance was measured at 593 nm using a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The standard curve was made with Trolox from 50 to 800 μmol/mL. The results were expressed as μmol Trolox equivalent per gram of extract (μmol TE/g of extract) [33 (link)].

The total polyphenol content was determined using the Folin–Ciocalteu assay described by Singleton et al. [36 (link)] with minor modifications. A quantity of 25 mg freeze-dried sample was dissolved in 25 mL water using 10 min of sonication and centrifuged at 5000 rpm for 10 min. A calibration curve was made with five calibration concentrations from 25 to 400 mg/L of gallic acid solutions in water. A volume of 0.1 mL of calibration or sample solution was mixed with 0.4 mL Folin–Ciocalteu reagent, 0.6 mL aqueous Na₂CO₃ 200 g/L, and 3.9 mL water in 15 mL centrifugal tubes shortly vortex mixed. After 2 h of dark incubation at room temperature, the absorbance was measured at 760 nm by a Genesys 150 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The polyphenol content was expressed as gallic acid equivalent (GAE) milligrams per gDM biomass.

The production of siderophores in five A. baumannii isolates that showed increased ability to excrete these molecules (CBIO024, CBIO086, CBIO117, CBIO123, and CBIO159) was evaluated in MM broth without iron, and in MM containing a low (22 µM) and a high (220 µM) concentration of FeCl₃·6H₂O. A bacterial inoculum was prepared by culturing the selected bacteria on nutrient agar for 24 h at 28 °C. The cell mass was then washed twice with NaCl (0.85% w/v) by centrifugation for 5 min at 4430×g (Hermle, centrifuge Z 326 k, Germany), removing the supernatant between washes. The resulting cell pellet was resuspended in MM broth supplemented with each of the FeCl₃·6H₂O concentrations and adjusted to a final OD of 2.0 at 600 nm (Thermo Fisher Scientific, Genesys 150 spectrophotometer, USA). Next, a 0.5 mL aliquot of each bacterial inoculum was added to Falcon tubes (15 mL) containing 4.5 mL of the MM broth without iron or supplemented with FeCl₃·6H₂O, until a final OD of 0.2 at 600 nm was reached. Cell cultures were incubated at 28 °C under constant shaking at 180 rpm with an oscillation diameter of 10 mm for 72 h (Heidolph, Shakers Unimax 1010, Germany). Siderophore production was quantified using CAS assay as described above. Three biological replicates were performed in a series of 4 samples.

The production of siderophores in five A. baumannii isolates that showed increased ability to excrete these molecules (CBIO024, CBIO086, CBIO117, CBIO123, and CBIO159) was evaluated in MM broth without iron, and in MM containing a low (22 µM) and a high (220 µM) concentration of FeCl₃·6H₂O. A bacterial inoculum was prepared by culturing the selected bacteria on nutrient agar for 24 h at 28 °C. The cell mass was then washed twice with NaCl (0.85% w/v) by centrifugation for 5 min at 4430×g (Hermle, centrifuge Z 326 k, Germany), removing the supernatant between washes. The resulting cell pellet was resuspended in MM broth supplemented with each of the FeCl₃·6H₂O concentrations and adjusted to a final OD of 2.0 at 600 nm (Thermo Fisher Scientific, Genesys 150 spectrophotometer, USA). Next, a 0.5 mL aliquot of each bacterial inoculum was added to Falcon tubes (15 mL) containing 4.5 mL of the MM broth without iron or supplemented with FeCl₃·6H₂O, until a final OD of 0.2 at 600 nm was reached. Cell cultures were incubated at 28 °C under constant shaking at 180 rpm with an oscillation diameter of 10 mm for 72 h (Heidolph, Shakers Unimax 1010, Germany). Siderophore production was quantified using CAS assay as described above. Three biological replicates were performed in a series of 4 samples.