4.3.2. Antibacterial Studies

The antibacterial effect of samples was investigated by the antibacterial disc diffusion test [46,47], using unsubstituted HA as a control. The samples were sterilized in a heating oven for 2 h at 160 °C. Then, 20 µL of LB-media containing Escherichia coli or Staphylococcus carnosus at an optical density of 0.015 (OD₆₀₀) were spread homogeneously on agar plates. Then, HA disc samples were placed on these agar plates and incubated at 37 °C for 24 h. The zones of inhibition for each plate were measured after 24 h of incubation (each test was performed in triplicate).

The effect of ion release from substituted HA powdered samples was examined via an indirect method [48]. The powdered samples were immersed in phosphate-buffered saline (PBS) for 24 h, and the supernatant extract was co-cultured with bacteria. The optical density (OD) of this medium was measured and reported in terms of turbidity. Higher optical density values were associated with greater turbidity of the bacterial suspension developed from bacterial growth during the incubation course.