Agar Well Diffusion Method

The studies were carried out by following standard procedures [28, 29]. Two types of agar, Agar Agar type1 (4 g) was mixed with Muller Hinton Agar (8 g) using 25 ml doubly distilled water and stirred well, placed in a microwave oven for 5 min. The mixture was turned to be viscous. It was transferred into petri dishes. After the plate gets cooled, each petri plate was divided into six parts. Wells (5 mm) were made on the agar plate using a cork borer under sterile conditions. The selected bacteria were swabbed on to the agar plate and the samples (20 µl) were injected into the specific wells using micropipette under sterile conditions.

The bacterial suspension (100 µL) (~ 104 and ~ 10 colony-forming unit (CFU/mL) of selected pathogenic bacteria, respectively) was spread in the agar plates. They were cultured in LB (Luria Bertani). Sterile paper disks (5 mm) were filled into the colloidal samples and kept on the agar plates. Disks were impregnated with gentamycin was also placed on nutrient agar, for positive controls also disk’s were impregnated. The incubation period of 24 h (at 37 °C) were observed for the plates. Zone of inhibition around the disks were then recorded.