Double beam spectrophotometer

Ultraviolet and visible spectra were recorded at UV-visible 1800 SHIMADZU double beam spectrophotometer. One milligram of AgNPs was suspended in 100 mL of distilled water. Distilled water was used as a blank and reference solution as well. UV-visible spectra were recorded in the scanning range of 250 nm to 600 nm using 3 mL quartz covets.

DPPH free radical scavenging activity of cow and buffalo cheddar cheese was determined by following the method of [23 (link)]. 1 ml sample was mixed with 1 ml DPPH solution (1 mM in methanol) and incubated at room temperature for 30 min. The absorbance was measured on a double beam spectrophotometer (Shimadzu, Japan). DPPH free radical scavenging activity was determined at 0, 40, 80 and 120 days of ripening and expressed as %inhibition.

The minimum inhibitory concentration of the extract was used to test its efficacy against biofilm formation. Quantitative assessment of the biofilm formations of E. faecalis and C. albicans and as a dual-species was carried out using the crystal violet (CV) assay in a microtiter plate. Briefly, fresh tryptone soya broth (100 μL) having MIC of the medicament was seeded with 10 μL of E. faecalis or C. albicans and each 5 μL of E. faecalis plus C. albicans in a 96-well microtiter plate. The plates were incubated at 37°C for 24 hours. After incubation, the planktonic cells were aspirated, and the wells were washed with sterile water to remove the free-floating bacteria. The adhering biofilm was fixed by heating and then stained with 0.1% crystal violet.
The wells were washed with sterile water and dried. The amount of biofilm formed was quantified by the addition of 200 μL of 95% ethanol/acetic acid mixture (1:1), and the optical density (OD) of the resulting solution was measured at 595 nm in a double beam spectrophotometer (Shimadzu, Japan). Background absorbance was compensated for by subtracting the OD of the sterile broth (negative control). The experiment was carried out in triplicate, and the mean OD was considered.

The elemental analyses of the synthesized silver(I) complexes for carbon, hydrogen, and nitrogen were performed by the Microanalytical Laboratory, Faculty of Chemistry, University of Belgrade. The ¹H and ¹³C NMR spectra of the N-heterocyclic ligands and the silver(I) complexes were recorded at room temperature on a Varian Gemini 2000 spectrometer (¹H at 200 MHz, ¹³C at 50 MHz). 5.0 mg of each compound was dissolved in 0.6 mL of DMSO-d₆ and transferred into a 5 mm NMR tube. Chemical shifts, δ, are reported in parts per million (ppm) and scalar couplings (J) are reported in Hertz. Chemical shifts were calibrated relative to those of the solvent. The abbreviations for the peak multiplicities are as follows: s (singlet), d (doublet), dd (doublet of doublets), and m (multiplet). In order to investigate the solution behavior of silver(I) complexes, the ¹H NMR spectra were recorded immediately after their dissolution and after 48 h standing in the dark at room temperature. The UV-Vis spectra were recorded on a Shimadzu double-beam spectrophotometer after dissolving the corresponding silver(I) complex in DMSO over the wavelength range of 200–900 nm. The concentration of the silver(I) complexes was 2.36·10⁻⁴ (1), 4.31^.10⁻⁵ (2), and 1.50^.10⁻⁵ M (3). The IR spectra were recorded as KBr pellets on a PerkinElmer Spectrum 100 spectrometer over the wavenumber range of 450–4000 cm⁻¹.