Viability assay of classical O395.

<p>O395 cells from mono- and co- cultures were sorted and stained with the dye JC-1 and analyzed by flow cytometry. Cells treated with CCCP for membrane depolarization was used as negative controls.</p

Growth of <i>V. cholerae</i> classical O395 in monocultures and cocultures.

<p>The strain O395 was grown individually in monocultures or cocultured with El Tor N16961 (1∶1) and CFU of O395 was determined at regular intervals. Data is represented as means ± SD, n = 4.</p

Reduction of CFU of classical biotype in cocultures is dependent on growth phase and pH.

<p><b>A.</b> Fold change in CFU of O395 at 9 h after mixing of (a) cultures of O395 and N16961 in logarithmic phase of growth, (b) logarithmic phase culture of O395 and late stationary phase culture (24 h) of N16961 (c) late stationary phase culture of O395 and logarithmic phase culture of N16961 and (d) late stationary phase cultures of O395 and N16961. Data is represented as means ± SD, n = 3. <b>B.</b> Classical O395 and El Tor N16961 were inoculated into standard LB medium or LB buffered to pH 7 and CFU of O395 was assayed at regular intervals. The experiment was repeated twice (ND <10⁴ CFU/ml).</p

Delayed loss of culturability of <i>O395</i>Δ<i>rpos</i> in cocultures with El Tor N16961.

<p>A. Growth of O395?<i>rpos</i> and O395 in cocultures with El Tor N16961. Data is represented as means ± SD, n = 3. B. O395Δ<i>rpos</i> and El Tor N16961 were grown separately for 24 hours and mixed in the ratio of about 1∶1. Samples were removed at the time of mixing (0 h), 36 hours and 48 hours after mixing and processed for confocal microscopy.</p

Lysis of classical O395 when grown individually in monocultures or in cocultures with El Tor N16961 Δ<i>dns</i>Δ<i>xds</i> (DNase⁻).

<p>CFU of O395 and O395 DNA in the supernatant (sup) were estimated in monocultures (a) and cocultures (b). DNA released into the supernatant when CFU of O395 in individual cultures decreased approximately six fold and that in cocultures decreased >10000 fold is indicated. Data is represented as means ± SD, n = 3.</p

Flow cytometric analysis of cocultures.

<p>N16961 and GFP labeled O395 were grown separately for 24 hours, mixed (1∶1) and samples were removed from the cocultures for flow cytometric analyses at 24 hours (A), 48 hours (B) and 7 days (C) after mixing. D. The GFP labeled (b) and non- labeled cells (c) from the cocultures were separated by FACS at the time of mixing (0 h) and 24 hour after mixing, and CFU of each population was determined. O395 monocultures (a) were used as an experimental control. Plating efficiency was scored as CFU per particle sorted from each gated population. Data is represented as means ± SD, n = 3.</p

Relative proportion of classical O395 and El Tor N16961 in cocultures.

<p>GFP labeled O395 and DsRed labeled N16961 were cocultured and relative proportion of the two biotypes was determined by confocal microscopy at the time of mixing (0 h) and 24 hour after mixing. CFU of O395 and N16961 at 0 h and 24 h is indicated (ND <10⁴ CFU/ml).</p