2.1.3. Antimicrobial activity assay In vitro antimicrobial activity of synthesized ligands was evaluated by agar disc diffusion method [40]. Mueller Hinton Agar (MHA) media (HIMEDIA, India) was used as a control medium for testing against bacteria and Potato Dextrose Agar (PDA) media (HIMEDIA, India) was used for fungal strain. After preparation, the MHA and PDA medias were incubated for 24 h and contaminations were checked. After incubation, the test organism was inoculated using sterile cotton bar on media. The sample discs were put gently on pre-inoculated agar plates and aerobically incubated for 24 h at 37 °C for antibacterial and for 48 h at 26 °C for antifungal assay. Dimethyl sulfoxide (DMSO) was used as control. Each disc was loaded with 25 µL of sample solution in DMSO containing 300 µg of synthesized compounds. 10 µL of ceftriaxone and amphotericin-B solutions containing 50 µg each in DMSO were loaded per disc for antibacterial and antifungal assays as positive control, respectively. The diameter of the inhibition zones in mm circling the disc were measured. Two gram-positive Staphylococcus aureus (cars-2) and Bacillus Megaterium (BTCC-18), two gram-negative Escherichia coli (carsgn-2) and Salmonella Typhi (K-323130) bacteria, and two fungal strains Trichoderma harzianum (carsm-2) and Aspergillus niger (carsm-3) were used in this study. We appreciate the recommendation, and this section is updated by changing the standard ciprofloxacin and miconazole with Ceftriaxone and Amphotericin-B. Also, the sentences of second part (4.8) are deleted and added to 2.3.1 as suggested by the reviewer. Antimicrobial activity using agar disc diffusion method In vitro sensitivities of two gram-positive and two gram-negative bacteria including two fungal strains against the synthesized compounds were evaluated by agar disc diffusion method. The formation of diameter of inhibition zones in mm by the synthesized analogues are shown in Table 5. Compound L2 showed moderate activity against Bacillus Megaterium bacteria while L1 showed promising antifungal activity against Aspergillus niger fungal strains compared to standard Amphotericin-B. Diameter of inhibition zones (mm) of the synthesized compounds, Ceftriaxone and Amphotericin-B against tested bacterial and fungal strains.
Section 4.8. There is no proper description of the results and no discussion in this part. Please revise - This sentence should be moved to the materials and methods or deleted from the section “‪Two gram-positive Staphylococcus aureus (cars-2) and Bacillus Megaterium (BTCC-18), two gram-negative Escherichia coli (carsgn-2) and Salmonella Typhi (JCM-1652) bacteria, and two fungal strains Trichoderma harzianum (carsm-2) and Aspergillus niger (carsm-3) ‪were used in this study.”