2.4. Determination of Minimum Inhibitory and Minimum Bactericidal Concentrations

The broth dilution method was employed to determine the minimum inhibitory concentration (MIC) of the crude plant extract. It was determined against the highly sensitive test strains of meat-borne bacterial pathogens such as S. aureus, E. coli, and Salmonella sp. The dried EtOH extract, aliquoted in phosphate-buffered saline (pH 7.2), was used as the test solution. The dosing range of plant extracts was computed by a factor of 2 (antilog 0.3) to obtain a final value ranging between 2000 and 32000 µg per mL in nutrient broth. Afterwards, each tube was inoculated with 100 µL of a fresh overnight culture of the appropriate bacterial isolate and incubated at 37°C for 24 hrs. Based on the preliminary experiments, the concentration range of the plant extract was further narrowed to obtain the specific MIC value (2000 and 32000 µg L). MICs were recorded as the lowest concentration that prevented the visible growth as indicated by the absence of turbidity in line with the control. To measure the minimum bactericidal concentration (MBC), MIC cultures were seeded (10 µL) on Mueller-Hinton agar and incubated for 24 hrs at 37°C. MBC was defined as the concentration which exhibited no growth of the colonies compared to the culture of the initial inoculum, of the same strain. Plant extract is considered as bactericidal if the ratio of MBC/MIC is ≤ 2, and otherwise as bacteriostatic. A ratio, ≥16, hints at the ineffectiveness of the extract [15].