Bacterial Load Enumeration in Infected Larvae

Changes to the bacterial load during infection or following treatment were determined via CFU enumeration. At each time-point, four larvae were randomly selected and individually homogenized in a lysing matrix tube containing six 1/8-inch metal beads in 800 μl of PBS-T, using a FastPrep F120 (MP Biomedicals, USA) at 6.0 m/s for 1 min. For CFU enumeration, serial ten-fold dilutions were plated on Middlebrook 7H11 agar, supplemented with 20 μg/ml of piperacillin (PIP, Sigma-Aldrich, UK) to inhibit the growth of native Gm flora [11 (link)]. PIP has no inhibitory activity on H37Rv (MIC = 320 μg/ml).