Effect of substrate availability on maintenance of infectivity.

<p><i>P. amoebophila</i> and <i>C. trachomatis</i> cells were harvested from infected amoeba and HeLa 229 cell cultures, respectively, and incubated for indicated periods of time in different host-free media. Subsequently, incubated bacteria were used to infect amoebae (<i>P. amoebophila</i>) or HeLa 229 cells (<i>C. trachomatis</i>), which were then fixed at 48 h or 24 h p.i., respectively. Bacteria were detected by FISH (<i>P. amoebophila</i>) or immunostaining (<i>C. trachomatis</i>). The observed infectivity, relative to that observed for 2 h incubation in DGM-D (<i>P. amoebophila</i>) or 30 min incubation in DGM-D6P (<i>C. trachomatis</i>) is depicted in (A) and (C), respectively. Data represent means and standard deviations from at least three replicate host-free incubations. For each sample a minimum of 600 amoebae (A) or 300 HeLa 229 cells (C) was counted. Statistically significant differences compared to the values obtained for DGM-D (A) or DGM-D6P (C) are indicated (ANOVA; ***, p≤0.001; **, p≤0.01; *, p≤0.05). In (B) representative fluorescence and DIC images of amoebae infected with <i>P. amoebophila</i> after 48 h host-free incubation in the indicated media are shown (FISH, red). The bar indicates 10 µm. In (D) representative confocal fluorescence images of HeLa 229 cells infected with <i>C. trachomatis</i> after 2 h host-free incubation in the indicated media are shown. Bacteria were detected by immunostaining (red), host cells and DNA were stained using HCS cytoplasmic stain (grey) and DAPI (blue), respectively. The bar indicates 25 µm.