4.1. Artificial Sputum Medium and Growth Conditions

ASM was prepared from sterile stock solutions, with the final concentrations shown in Table 1. The mucin stock solution (5% (w/v)) was prepared with deionized water sterilized by autoclaving at 121 °C for 30 min. Salmon testis DNA and ferritin were dissolved in sterile deionized water. All other stock solutions were prepared and sterilized using 0.22 µm-pore syringe filters.

ASM components and their final concentrations.

ASM recipe is modified from Sriramulu et al. (2005) and Quinn et al. (2015) [22,29]. * Concentration of NaCl and NaHCO₃ are varied depending on the ASM conditions (Table 2).

Artificial sputum media were prepared with and without HCO₃⁻ (Table 2). Please note that the ASM used in the flow cytometry experiments did not contain DNA because SYTO9 and PI, being nucleic acid stains, would bind to extracellular DNA.

ASM conditions and modifications.

The pH of the NaHCO₃-free ASM was adjusted by mixing appropriate volumes of the HEPES acid and Na-HEPES stock solutions. For the NaHCO₃-containing ASMs, the components of NaCl were reduced to maintain isosmotic conditions. Thus the NaCl concentration was reduced to 75 mM in the ASM containing 25 mM NaHCO₃, and to zero in the ASM containing 100 mM NaHCO₃. Both of the NaHCO₃-containing ASMs were incubated in 5% CO₂ giving pH 7.4 and 8.0, respectively. Since NaHCO₃ is heat-sensitive, 0.22 µm-pore syringe filters were used to sterilize the NaHCO₃ stock solution. Furthermore, NaHCO₃ was added to each ASM immediately prior to bacterial inoculation.