4.9. Determination of Antibacterial Activity of Lipid Extracts

The antibacterial activity of lipid extracts was tested against two bacterial species, a Gram-positive and a Gram-negative, specifically Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 25922, respectively. The inoculum for the antibacterial tests was obtained by growing the bacteria in tryptic soy broth (TSB) at 37 °C and 150 rpm overnight (approx. 18 h). Afterward, the culture was diluted in 0.9% NaCl solution and adjusted to a concentration of 10⁸ colony-forming units (CFUs) per mL. The assays were performed in 96-well plates and in triplicate. Lipid extracts in DMSO were mixed with Mueller Hinton Broth and distributed 150 µL per well to obtain the final concentrations of 20, 60, 125, and 200 µg mL⁻¹. Each well was then inoculated with 50 µL of the bacterial suspension. Two controls were included, namely, a bacterial inoculum only and a control containing the bacterial inoculum and the same amount of DMSO added to the bacterial culture with lipid extracts. The bacterial suspensions were incubated at 37 °C and 120 rpm for 24 h. After incubation, bacterial cultures were serially diluted in 0.9% NaCl and plated in Mueller Hinton agar using the drop plate method. After incubation at 37 °C for 18 h, viable counts were determined as log CFU mL⁻¹ and compared with the control with DMSO. Extracts were considered to have bacteriostatic or bactericidal effects if a decrease of <3-log and ≥3-log in CFU mL⁻¹, respectively, was observed.