2.4.1. Antibacterial and antifungal activity
In this research, the antibacterial potential of the extracts was evaluated against four types of bacterial species and two types of fungus strains. Gram-positive bacteria were Staphylococcus aureus (ATTCC6538), (Bacillus subtilis (ATCC6633), and Gram-negative bacteria were Escherichia coli (ATCC6538), Pseudomonas aeruginosa (ATCC 9027). Also, Aspergillus oryzae, and Candida albicans (MTCC 227) fungus were used. At 37°C, Mueller Hinton agar medium was used to culture bacteria for 24 h. The fungal samples were cultured in Staphylococcus aureus and PDA medium at 29 ° C for 72 h.
i. Disk diffusion test
The disk diffusion technique was utilized to evaluate the extract’s antibacterial activity and CuO NPs (Alvand et al. 2019). On the Muller Hinton Agar medium, 0.1 mL of the diluted solution of each organism was evenly distributed, and the results were compared with DMSO, as a negative control. Three sample concentrations (6.25, 12.50, and 25 mg/mL) were tested against the microorganisms in DMSO. A Filter paper disc with an internal diameter of 6 mm was treated with the samples and located on the inoculated plates for 24 h at 37°C. The antibacterial activity of the samples was determined by measuring the inhibitory area around each disk. In control tests, amoxicillin (25 mg/disk), kanamycin (10 mg/disk), and cephalexin (30 mg/disk) were employed as conventional antibiotics. As a negative control, the blank disks were used and loaded with 100 mL of the solvent.
ii. MIC& MBC test
The antimicrobial activity of the samples was also investigated using the MIC tests (Montazerozohori et al. 2014). In this technique, several samples with a certain concentration range (0.0024-12.5 mg/mL in this research) were prepared in sterilized conditions and a specific volume of Muller Hinton Broth and microorganism solution (0.65 mL and 0.1 mL, respectively) were mixed to each tube, which contained 0.25 mL of the sample. It was incubated for 24 h, 37°C, and the sample's lowest concentration at which the microorganisms' growth was inhibited was recorded as the MIC. In MBC screening, the Muller Hinton Agar medium was coated with a certain amount of the diluted sample (0.0024-12.5 mg/mL), and following which, it was incubated for 24 hours at 37°C. The lowest concentration samples, which caused the least growth of microorganism, was reported as MBC (Bauer et al. 1966).
iii. Calculations of the quantities of nucleic acids and proteins
Firstly, 10 mL of high-quality Mueller Hinton Broth (MHB) (OD600 = 0.4) was suspended in the fresh bacterial culture. 100 µL of the obtained suspension was mixed with the previously prepared samples with concentrations at the MIC. As a control experiment, the tubes were shaken at 250 rpm while being incubated at 37°C at intervals of one hour using sterilized distilled water. (1–6 h). The collected samples were centrifuged for five minutes at 4000 rpm. Finally, the samples’ optical density (OD) was measured at 260 and 280 nm, respectively (Nejabatdoust et al. 2020).
iv. Time-kill and growth inhibition curves
The antibacterial properties of the extracts and CuO NPs were assessed using time-kill curves on Gram-negative and Gram-positive bacteria. Concentrations equal to the MIC of the samples were prepared. An inoculum size of 4.5–5.5 ×105 CFU/mL was added and treatment for 1, 2, 3, 4, 5, and 6 h incubated at 37°C. A typical saline solution (0.085%) was used during the serial dilution procedure. Then each tube had portions of 100 µL removed, and a colony counting unit was used.
Calculations for bacterial viability and inhibition were made as follows:
Percentage of viability = \(\frac{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{o}\text{l}\text{o}\text{n}\text{i}\text{e}\text{s} \text{c}\text{o}\text{u}\text{n}\text{t}\text{e}\text{d} }{\text{N}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l} \text{c}\text{o}\text{l}\text{o}\text{n}\text{i}\text{e}\text{s} }\)×100
In this experiment, the control wells comprised only bacteria and Mueller-Hinton broth (Alvand et al. 2022).
v. Electron microscopy test
The morphological changes were assessed using Scanning Electron Microscopy (SEM). The cells were rinsed twice in PBS and allowed to settle overnight in 2.5% glutaraldehyde. Various ethanol concentrations (60%, 70%, 80%, 90%, and 100%) were used to dehydrate the samples. In a sputter coater, dried cell samples were coated with gold and meticulously examined using a scanning electron microscope (Kakishita et al. 2021).