Uv visible spectrophotometer

After 21 days of co‐culture, the samples were rinsed three times with PBS, then fixed in 4% paraformaldehyde for 30 min, and finally the fixed samples were stained with ARS dye for 30 min. The stained samples were rinsed three times with deionized water to remove excess ARS dye. Images of the stained samples were obtained using fluorescence inverted microscopy. For further quantitative analysis of calcium deposition, the stained samples were reacted with 10% cetylpyridinium chloride (Sigma‐Aldrich, USA) for 1 h at room temperature, and the supernatant was taken in a 96‐well plate, and the absorbance at 562 nm was measured by UV–visible spectrophotometer (Perkin Elmer, USA).

In vitro biodegradation of cypermethrin was performed in 100 ml flask with 50 ml minimal salt medium, supplemented with 50 ppm cypermethrin and inoculated with 1 ml SG2 culture before optimization the biodegradation conditions. Bacterial inoculum was prepared by inoculating single colony in 50 ml of MSM. Inoculated flask was incubated in an incubator shaker @ 100 rpm at 30 °C. Bacterial cells from the log phase, harvested by centrifugation (5000 rpm for 10 min) and washed with minimal medium were used for the subsequent studies. Uninoculated medium acted as control. Bacterial growth was monitored by taking absorbance using UV–Visible spectrophotometer (Perkin Elmer) and the residual cypermethrin concentration was measured by HPLC (Dionex), after extraction according to Anastassiades et al. (2003 (link)).

Antioxidant molecules were extracted from 1 g of ground sample, using 5 mL of a methanol/water (70:30 v/v) solution, kept under motion for 60 min at room temperature, and then paper filtered. The solvent was evaporated, and the extract was then dissolved in 10 mL of methanol, thus centrifuged at 5040× g for 15 min at 4 °C. Analyses were performed in triplicate mixing 100 μL of surnatant and 1 mL of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) methanolic solution (0.2 mm), bringing the mix to a final volume of 2.4 mL with methanol. The absorbance of the solution was recorded at 517 nm by a Perkin Elmer UV-Visible spectrophotometer after an incubation time of 30 min in the dark at room temperature. Blank was prepared and analyzed following the same procedure, using 100 μL of methanol instead of sample. The radical scavenging activity was calculated as follows:
where Abs0 was the absorbance of the blank and Abs1 was the absorbance of the sample. The Trolox Equivalent Antioxidant Capacity value (TEAC) expressed as μmol Trolox equivalents/gram of dry weight (μmol TE/g of dw) of the samples was calculated from the calibration curve obtained by measuring the absorbance at 517 nm of Trolox methanolic solutions at different concentrations. Three replicates were analyzed for each treatment condition.