Sterile disc

The BEO was evaluated for its in-vitro antimicrobial activity as per Sfeir et al. [31 (link)] against eight pathogens and spoilage organisms by Kirby-Bauer disc diffusion method on Mueller-Hinton agar (MHA) plate. Discs were prepared by incorporating 20 µL of BEO of different concentrations (2.5%, 5%, 7.5% and 10%) into each sterile disc (HiMedia, Mumbai, India) separately. Each test organism’s inoculum was prepared in sterile MHA and the turbidity of each suspension was adjusted equal to 0.5 MacFarland standard, which is equal to 1.5 × 10⁸ colony forming units (CFU/mL) for bacteria. Each bacterial culture (20 µL) used in this study was spread on separate MHA plates and the discs containing the above four concentrations of BEO were placed on each MHA plate. The plates were incubated at 37 °C for 18–20 h and the zone of inhibition (ZOI) were measured (in cm) by measuring the diameters of clear zones around the discs. Streptomycin filter discs were used as a control in the study and the ZOI of the BEO was compared with that of the control drug against each bacterium. Each test was performed in triplicate, and the results are expressed in cm ± standard deviation (SD).

Antimicrobial activity of active fraction was assessed by disk diffusion method against Staphylococcus epidermidis (MTCC 435), Staphylococcus aureus (MTCC 740), Bacillus cereus (MTCC 1272), Escherichia coli (MTCC 40), Klebsiella pneumoniae (MTCC 661), Micrococcus luteus (MTCC 7950) Aspergillus flavus (MTCC 2590), Fusarium moniliforme (MTCC 6576), Bipolaris maydis and Alternaria alternata (MTCC 1362). The sterile discs (6 mm, Himedia) were impregnated with 30 μL of crude extract. The pathogens were inoculated in Mueller-Hinton broth (24 h for bacteria, Himedia) and Sabouraud Dextrose both (72 h for fungi, Himedia). The well-grown bacterial and fungal cultures were plated on Mueller-Hinton agar and Potato Dextrose agar respectively (Himedia). sterile discs loaded with extract were placed on the plate. Chloramphenicol discs (Himedia) were used as positive control for antibacterial assay, while nystatin discs (Himedia) were used for the antifungal assay. Discs impregnated with dimethyl sulfoxide (DMSO, Himedia) were used as the solvent control. The plates were incubated at 37 °C and room temperature (for test bacteria and fungi respectively) and the zone of inhibition was measured.

Mueller−Hinton agar (Himedia Laboratories Pvt, Ltd., Mumbai, India) was the agar selected for the antibiograms. The initial inoculation level reached approximately 150 × 10⁶ CFU/mL (colony-forming units) (0.5 McFarland scale) for all strains tested.
The method applied was the agar diffusion method. Briefly, 1 mL of inoculum from each strain was plated on the surface of Mueller−Hinton agar, and the antibiotic discs were placed as well.
Levofloxacin (5.03 BCg/disc) for E. coli, penicillin-G (10 units/disc) for S. aureus strains, and vancomycin (30 μg/disc) for Streptococcus strains were employed as reference antibiotics; 10 μL of each extract were placed on sterile discs (Himedia Laboratories Pvt, Ltd., Mumbai, India), and petri dishes were incubated overnight at 37 °C. The inhibition diameter around the disc was measured with a Traceable Carbon Fiber Digital Caliper (Fisher Scientific Ltd., Nepean, ON, Canada). All measurements were performed in triplicate (n = 3).