FIG. 4.

Restoration of the culturability of VBNC bacteria. Legionella suspensions were analyzed for living and metabolically active cells by using the TVC labeling kit as described in Materials and Methods. The volume of each cell suspension containing 10⁶ CFU/ml viable L. pneumophila was determined. A. polyphaga (10⁵ CFU per well in 24-well plates) was infected with 10⁶ CFU/ml (multiplicity of infection, 10) of viable L. pneumophila (measured by TVC). After 1 h of incubation at 37°C, the monolayers were washed, incubated for 5 days, and then lysed with 0.05% Triton X-100. Aliquots were plated on BCYE agar for colony counting and were quantified by real-time PCR and immunodetection. Legionella viability could not be measured on the samples, because viable amoebae could be labeled at the same time, making the reading difficult. Control chlorinated bacteria (treated cells) incubated in PAS buffer without A. polyphaga remained culture negative. The results are expressed as described in the legend to Fig. 1. T, time.