Rabbit Red Blood Cells Haemolysis Assay

The haemolytic activity of MRSA was assessed using a functional test that measures the release of hemoglobin from erythrocytes. MRSA (10⁶ CFU/ml) was grown at 37°C in a shaking bath in the absence or presence of the compounds. After 16 h, the cultures were centrifuged (6,000 × g 4°C), and the supernatants were sterile filtered (0.22 μm). 100 μL were added to 900 μl haemolysin buffer (0.145 M NaCl, 0.02 CaCl₂) and 25 μl of defibrinated rabbit blood. The samples were incubated in an orbital shaker at 37°C for 1 h. Then the samples were centrifuged (5,500 × g, room temperature, 1 min) to pellet intact erythrocytes, and the supernatants were transferred to a 96 well plate to measure absorbance at 541 nm. Sterile culture medium was used as the reference for 0% haemolysis, and a bacterial culture supernatant devoid of any treatment served as the standard for 100% haemolysis. The percentage of haemolysis inhibition was calculated over the control cultures. All the assays were performed at least twice with triplicate determinations for each condition.