Test agar ph 8

Impregnated allografts were immersed in 2 mL phosphate-buffered saline (PBS, pH 7.4, Sigma-Aldrich, Schnelldorf, Germany), vortexed for 1 min, and incubated at 37 °C. After 1, 2, 3, 4, 5, 6, and 7 days, the elusion was completely removed and stored at −20 °C until release kinetic measurement. Fresh PBS was added for the next measurement. Release kinetic measurement was performed using a conventional microbiological agar diffusion assay with Bacillus subtilis (in Test Agar pH 8.0, Merck KGaA, Darmstadt, Germany). A total of 100 µL of each elusion or 10-fold dilutions (from 10.000 to 0.01 mg/L) of each antibiotic was added in a central hole (diameter: 6 mm). Plates were incubated at 37 °C under aerobic conditions. After 24 h, the inhibition zone was measured for each plate (in cm). A standard curve was obtained by logarithmic regression and used to predict the concentration of each antibiotic for each elution. The experiments were carried out in triplicates.

Bacillus subtilis (Merck KGaA, Germany in Test Agar pH 8.0 Merck KGaA, Germany), Staphylococcus aureus ATCC 29213 (American Type Culture Collection, LGC Standards GmbH, Wesel, Germany) and methicillin-resistant Staphylococcus aureus MRSA DSM 46320 (Leibniz Institute DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen— German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were used for antibiotic delivery and antibiotic susceptibility assays.