3.5.1. Agar Well-Diffusion Assay

The antibacterial activity of the extracts was tested against Staphylococcus aureus (ATCC 6358) and Escherichia coli (ATCC 25923) using the agar well-diffusion method. Extracts (500 mg/mL in DMSO) were separately sterilized by filtration using 0.22-µm membrane filters (Indiamart, Vadodara, India). The surface of the Muller–Hinton agar plate was infected by spreading a volume of microbial inoculum across the entire agar surface. Then, a sterile cork borer was used to aseptically punch a hole with a diameter of 6–8 mm, and each extract (100 µL) was added to individual wells. Agar plates were incubated under the appropriate conditions for the test microorganism [50]. Standard doxycycline and amikacin antibiotics were used as controls. DMSO was used as a negative control that showed no bacterial inhibition.