Fastprep 24 5g homogeniser

Dissection and infection of pig lungs were performed as described in Harrison et al. [9] .
Briefly, pig bronchioles were dissected, UV sterilised and transferred to 24-well plates with 400 µL of 0.8% agarose/ASM (Artificial Sputum Medium [14] ) as a pad. Bronchiole tissues were infected with the bacterial isolates using a syringe and 500 µL of ASM were added to each well. Uninfected bronchiole tissues were used as negative controls. Plates were then covered with UV sterilised breathable membranes (Sigma Aldrich) and incubated at 37°C for 7 days. Tissues were then washed in 500 µL of PBS, transferred to sterile bead tubes containing 1 gm of metal beads (2.4 mm, Fisher Scientific) and 1 mL of PBS, and homogenised using a FastPrep-24™ 5G homogeniser (MP Biomedicals) for 40 sec at 4.0 m/sec. Biofilm homogenate was transferred to 96-well plates, serially diluted and plated on LB agar plates for calculating the bacterial load.
Replicate infected tissues were treated with antibiotics by transferring the washed infected tissues to 48-well plates containing 300 µL of either MHB or ASM containing ciprofloxacin (Thermo Fisher), meropenem (Sigma), tobramycin (Thermo Fisher) or combination therapy at the specified concentrations and incubated at 37°C for 24 hours. Antibiotic-treated tissues were then washed, homogenised and plated as previously described.