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Processes 2020, 8, 388 4 of 14 TSB and then kept incubator at 37 ◦C for 24 h, and optical density was measured at 550 nm. The lowest concentration of AgNPs which restricted the 99 % of bacterial growth was considered as MIC. Further to determine the MBC value, 100 μL bacterial inoculums were streaked on the agar plate from the MIC tubes and incubated at 37 ◦C for overnight. The lowest concentration of Ns-AgNps at which no bacterial growth was seen on inoculated agar plate has been considered as MBC. 2.7. Detection of Anti-Biofilm Activity The anti-biofilm activity of biosynthesized Ns-AgNPs was observed against the biofilm producing standard bacterial strains with or without Ns-AgNPs. Biofilm production of the concerned pathogenic bacteria was carried out using the tissue culture plate (TCP) method as explained by Balasamy et al. [20], which is commonly applied worldwide and considered as standard test for detection of biofilm production with some modifications. MTCC bacterial strains of K. pneumonia, E. coli, S. aureus, P. aeruginosa, and E. faecalis were grown on blood agar plate for overnight at 37 ◦C and the next day a single colony of all bacteria were inoculated into different conical flasks containing the 100 mL TSB at 37 ◦C for 6–7 h with shaking at 100 rpm until to obtain approximately 2.5 × 108 CFUs/mL. These bacterial inoculums were further diluted (1:100) with fresh TSB medium to approximately 106 CFUs/mL and transferred it to 96 wells flat bottom TCPs and incubated at 37 ◦C for overnight. After completing the incubation, the old culture medium was changed with a fresh TSB medium including different concentrations of Ns-AgNPs (6.5–100 μg/mL), without disrupting the biofilm. Samples with AgNPs were incubated further at 37 ◦C for 24 h. Thereafter, the medium was discarded; each well were gently washed two times with sterile 1x Phosphate buffer saline (PBS) to get rid of the planktonic state or free floating bacteria and dried it at room temperature for 20 min. 0.1% crystal violet solution was added to each well for 15 min to stain the biofilms. The surplus stain was eliminated by washing three times with sterile 1% PBS, and dried at room temperature for 30 min. 100 mL of 95% ethanol was then added to each well. Optical densities (OD) of stained acquired biofilm were observed by a micro ELISA reader at wavelength 595 nm. Experiments were repeated in triplicate. Average OD values of sterile medium were measured and deducted from the all test values. [21]. The OD of sample was converted to percentage of biofilm inhibition, calculated as follows: Percentage of biofilm = OD of the test/OD of the control × 100 2.8. Determination of In Vitro Anticancer Activity of Synthesized AgNPs 2.8.1. Cell Culture and Cell Line Maintenance Breast cancer cell line (HCC712) was acquired from National Centre for Cell Science (NCCS), Pune, India. The HCC712 cells were freshly cultivated as monolayer in Dulbecco’s Modified Eagles’s Medium (DMEM), supplemented with 10% FBS, 1% glutamine, and 100 U/mL penicillin/streptomycin and incubated at 37 ◦C in 5% CO2 atmosphere. It was grown in a 75 cm2 tissue culture flask. 2.8.2. Cell Viability Test The colorimetric MTT assay was applied to scrutinize the cytotoxic effects. The activity of Ns-AgNPs was examined against HCC712 cell lines (1 × 106 cells/mL) and the culture was seeded in 96 flat-bottom well plate that was suitable for high throughput screening. Different concentrations (25 to 200 μg/mL) of silver nanoparticles were added to the cultures and incubated for 24 h in 5% CO2 atmosphere. After 24 h, the cells were washed with PBS followed by mixing of 100 μL of MTT, and the cells were again incubated for 3–4 hr at 37 ◦C in 5% CO2 atmosphere. The formazan crystals were suspended in 200 μL of DMSO and optical density of each well was observed. The quantity of formazan product as measured in the calorimetric assay at 570 nm with a reference filter at 630 nm determined and the growth inhibition rates were calculated as a percentage. Growth inhibition = A570 of treated cells/A570 of control cells × 100.PDF Image | Silver Nanoparticles Seed Extract Nigella sativa blackseed
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