2.4. In Vitro Antibacterial Activity and MIC Determination

Klebsiella oxytoca ATCC 51983 & Escherichia coli ATCC 35218 & Staphylococcus aureus ATCC 25923, and Bacillus cereus ATTC 11778 were purchased from the American Type Culture Collection (ATCC) and were chosen for the antibacterial screening. In addition, bactericidal activity was validated utilizing known antibiotics, namely ciprofloxacin [39]. An agar-well diffusion method was applied to determine antibacterial properties [40]. Firstly, 100 µL of preserved bacteria were transferred into nutrient agar plates, distributed by streaking, and incubated at 37 ± 2 °C for 24 h [41]. Afterward, 20 μL of the tested CMC/Ag-NPs (2 mg/mL) were added to each well and left in the refrigerator for 1 h. Then, they were incubated overnight at 37 ± 2 °C. After the incubation period, the diameters of clear inhibition zones, including the diameter of the disc (mm), were measured using a Vernier caliper [41]. The antibacterial activity of green CMC/Ag-NPs was tested in-vitro, using the agar well diffusion method [42]. Overnight cultures of each strain, at 0.5 McFarland standard, were spread onto Luria-Bertani (LB) plates, which were pierced with a 6 mm diameter cork borer and loaded with 50 µL of CMC/Ag-NPs diluted in 1% DMSO at various concentrations (25, 50, and 100 μg/mL, w/v). After incubation, the radius of the inhibition zone was measured with a Vernier caliper. Minimal inhibitory concentrations (MICs) against tested organisms were determined using the microbroth dilution method and resazurin dye [43]. In 96 well plates, micro-dilutions of overnight developed culture strains (McFarland turbidity of 0.5) were cultivated in Luria–Bertani broth. CMC/Ag-NPs, at various concentrations (50, 25, 12.5, 6.25, 3.12, and 1.56 μg/mL), were added, and plates were incubated overnight at 37 °C. Any shift in culture color from blue dye to pink dye inside viable cells was visually examined [44]. The MIC value was determined by determining the lowest concentration of CMC/Ag-NPs, at which point the dye color changed. The MBC value was calculated when no colony appeared after plating directly into the agar plate. Antibacterial activity was described using the MIC index (MBC/MIC). Bactericidal activity is indicated by a MIC index of 1–2, whereas bacteriostatic activity is indicated by a MIC index of 4–16 [43].