2.6.2. Percentage of Bacterial Growth Inhibition

The percentage of bacterial growth inhibition in the presence of the *C. pentandra*/Ag-NPs was evaluated quantitatively by following the method of Shameli et al. [20] with some modification. This analysis is based on spectrophotometrically OD value measurements to evaluate bacterial growth. The different masses of fibers (1, 2 and 4 mg) were placed into a sterile 96-well plate. The inoculum was prepared by subculturing 100 μL of stock culture bacteria into new sterile MHB. The inoculum was incubated overnight at 37 ◦C in the incubator. Afterward, the optical density OD600 of the bacterial suspension was adjusted to 1.0 absorbance using UV spectrophotometer (Secomam, Champigny sur Marne, France). This OD600 value corresponds to 8 <sup>×</sup> 10<sup>8</sup> CFU/mL. The adjusted inoculum was then diluted to 105 (1:1000 dilution) CFU/mL using sterile MHB. The volume of 100 μL of adjusted inoculum (10<sup>5</sup> CFU/mL) was added to each 96-well plate containing the *C. pentandra*/Ag-NPs. The final volume of each 96-well plate ensures 140 μL by adding sterile MHB. The inoculum of *C. pentandra*/Ag-NPs free medium under the same condition and volume was used as a blank control. The blank control of *C. pentandra*/Ag-NPs sample in MHB media in the absence of inoculum was used. The ampicillin standard (8 μg/mL) was used as positive controls. All the 96-well plates were then sealed using parafilm to avoid the evaporation from each well followed by incubating at 37 ◦C for 18 h. After the incubation process, the OD600 value of the inoculum in each well was determined using a microplate reader (Infinite 200, Tecan, Männedorf, Switzerland) at 600 nm wavelength. The data were reported in the mean ± standard error of the mean (S.E.) of three experiments. The percentage of bacterial growth inhibition rate was calculated using Equation (4) as below:

$$\text{Basterial growth in inhibition } \left( \% \right) = \left( \frac{OD\_{\text{control}} - OD\_{\text{sample}}}{OD\_{\text{control}}} \right) \times 100 \tag{4}$$

where, *ODcontrol* is inoculum only, and *ODsample* is the differences between *ODC. pentandra*/*Ag-NPs* with bacteria and *ODC. pentandra*/*Ag-NPs* without bacteria (blank).
