To evaluate the ability of PFA to kill the intracellular bacterial pathogens Burkholderia pseudomallei (Bp) strain 1026b and Francisella tularensis tularensis (Ft) strain Schu S4 during fixation of infected cells, we performed time course studies in which infected murine macrophages were treated with a range of concentrations of paraformaldehyde solution (0.5-4%). Murine BMDMs were infected with each of the pathogens at an MOI of 100 and cultured for 12-15 hrs to allow bacterial replication in infected cells. After the cells were harvested, washed and enumerated, they were distributed into O-ringed centrifuge tubes (5 x 105 cells/tube) and pelleted via centrifugation, supernatant aspirated away, and then the cells were resuspended in PFA solution (0.5, 1.0, 2.1, or 4.21%). Dilution plating was performed at each treatment time point (1, 5, 10, 15, 30, and 45 minutes as well as 1, 4, 8, and 24 hrs) to determine how many viable bacteria remained in each fixed cell sample (Table 5-1).
