2.8. Bacterial Inoculum Preparation for In Vivo Study

The S. epidermidis 103.1 strain was cultured as described above. The bacterial inoculum was introduced to the rats on pre-contaminated screws prepared immediately prior to each surgery. On the day of surgery, S. epidermidis overnight cultures were centrifuged (2500× g for 10 min), washed in phosphate buffered saline (PBS) twice and then adjusted to an optical density of 0.50 (±0.01) at 600 nm. The threaded portion of the screw was submerged and incubated statically at room temperature for 25 min. Test screws were inoculated in parallel within each series of experiments. A quantitative assessment of S. epidermidis adhesion to the test screw was performed by sonication in PBS (3 min) followed by serial dilution, plating on 5% horse blood agar (Oxoid) and incubation overnight at 37 °C. The target inoculum for each screw was 1.5 × 10⁶ CFU/screw (range: 0.9–2 × 10⁶ CFU/screw). All screws were implanted within 30 min of preparation.