2.3. Time–Kill Curves

Four milliliters of blank medium, 0.5 mL of 10-times the final drug concentration, and 0.5 mL of logarithmic M. hyopneumoniae were added to a bottle in turn and then mixed. The tilmicosin concentration in the culture system was in a certain range (1/2, 1, 2, 4, 8, 16, and 32 -times the MIC that was determined for an M. hyopneumoniae inoculum of 10⁶ CFU/mL). A growth control (not exposed to the drug) and a sterility control (medium at pH 7.7 without the drug or M. hyopneumoniae) were indispensable. Penicillin bottles were cultured for 60 h in the environments described above. Aliquots (100 μL) of the culture were taken from each bottle at 0, 1, 3, 6, 9, 12, 24, 36, 48 and 60 h to detect the M. hyopneumoniae population. After 7 days, the results were read using an inverted microscope (Leica, Weztlar, Germany).