Bullet blender blue

O. tsutsugamushi strain UT-76 (a Karp-like strain from Thailand) was propagated in T25 cell culture flasks containing a confluent monolayer of L929 cells which were grown in DMEM supplemented with 10% FBS at 35°C with 5% CO₂. After 7 days of infection the cells were sub-cultured onto fresh L-929 cell monolayers. The infected flasks were harvested by scraping using a sterile inoculating loop in 1ml spent medium and disrupted to release intracellular bacteria by lysing in a bullet blender (BBX24B, Bullet Blender Blue, Nextadvance, USA) used at power 8 for 1 min. The lysed bacteria were added to a T25 flask (100 μl/flask) containing an uninfected L-929 monolayer at 70–100% confluence. Infected flasks were passaged at a ratio of 1:5 (original culture flask: subculture flask). Bacteria were sub-cultured for a total of <20 passages, where passage 0 was the original clinical isolate.
Antibiotics were only used in the experiment testing the effect of antibiotic addition. Here antibiotics were used at the following concentrations: chloramphenicol 150 μg/ml, penicillin G 100 μg/ml, penicillin G + streptomycin (premix from Sigma-Aldrich) 125 μg/ml + 200 μg/ml.
Cell lines and bacteria were tested for the presence of mycoplasma and confirmed to be mycoplasma-free using the VenorGeM PCR detection kit (Minerva biolabs).

One third lungs were placed in a Bullet Blender Blue (Next Advance, Troy, NY) and homogenized at speed 8 for 4 minutes. Tissue homogenate was plated at a 1:5 dilution on 7H11 agar plates. The limit of detection is 15 CFUs for lungs.