Root canal bacteria in teeth with apical periodontitis were enumerated after extraction and incubation. Canals in 36 teeth were sampled after: S1, incubation for 2 hours (group A), 2 days (group B), 4 days (group C), and 6 days (group D); S2, subsequent incubation for 1 week; S3, canal disinfection; and S4, final incubation for 1 week. Bacterial concentrations were determined by culture (colony-forming unit [CFU]) and epifluorescence-microscopy (EFM) and compared by using pairwise and exact-permutation tests (p < 0.05). CFU counts were lower than EFM counts. CFU counts in S1 were higher in GpA than in GpC (p < 0.004) and GpD (p < 0.02). EFM counts in S1 were higher in GpA than in GpC (p < 0.02). Both enumeration methods showed bacterial counts decreasing from S1 to S2 (p < 0.04). EFM was superior to culture in this ex vivo model. The indigenous flora survived incubation for 6 days, but the adverse effect of initial access would preclude testing of disinfection protocols that require two sessions.