Burkholderia Select Agent Protocols

All Select Agent work was carried out in CDC/USDA Tier 1 select agent approved BSL-3 and ABSL-3 facilities at the University of Florida following Tier 1 regulations. All protocols were approved by the Institutional Biosafety Committee prior to implementation under numbers BA-4200 and RD-4158. B. mallei strains (China 7, G-2(3), India 86-567-2, Ivan, SAVP1, and Turkey #1; (obtained from the CDC)), B. pseudomallei 1026b (CDC/USDA registered in house bacterial inventory) and B. thailandensis strains (E555, obtained from Mahidol University; and TxDOH, obtained from CDC) were grown on LB Lennox broth or agar (5 g/L NaCl) (LB, Fisher BioReagents, Pittsburgh, PA, USA) and grown at 37 °C. LB broth was used for liquid growth of all strains. B. mallei strains were grown on or in LB media with the addition of 4% (v/v) glycerol (LB4G). Select Agent excluded strain Bp82 [45 (link)] and was grown on LB or TSA with 0.6 mM adenine (Amresco, Solon, OH, USA). The mouse cell line RAW264.7 (American Type Culture Collection, ATCC) was grown in Dulbecco’s Modified Eagle Medium (DMEM)-high glucose + l-glutamine (HyClone) with 10% FBS (HyClone) in 5% CO₂ at 37 °C. All plastic ware was Corningware with CellBIND surface. Culturing cells was carried out essentially as described previously [46 (link),47 (link),48 (link),49 ,50 (link),51 (link)].