Polystyrene culture tube

To assess the interaction between L. lactis and patient derived P. aeruginosa, L. lactis was co-cultured with P. aeruginosa in the presence of mucin (Fig. 1). A minimal mucin medium containing intact mucins was purified from porcine gastric mucin (PGM) (Sigma-Aldrich, St. Louis, MO) per protocol described by Flynn et al.^{15 (link)} PGM was first dialyzed and filtered to remove small metabolites that could potentially support growth of bacteria. L. lactis was introduced into 2 mL of mucin minimal medium in molten 1.0% agar at 50°C in a polystyrene culture tube (Fisher Scientific Company, Pittsburg, PA), under anaerobic conditions for a final concentration of 5 × 10⁵ CFU/mL (L. lactis did not grow under aerobic condition) and washed twice with phosphate buffered saline (PBS). Tubes without L. lactis were used as a negative control. After solidification of the L. lactis fraction, 1 mL of molten minimal medium 0.7% agar without mucins was introduced and then inoculated with 1 of the P. aeruginosa strains, as described in P. aeruginosa preparation above, for a final concentration of 1 × 10⁶ CFU/mL. After solidification, co-cultures were incubated at 37°C for 72 hours. The upper agar plugs were removed and homogenized in a 1 to 10 dilution with PBS. Values of CFU/mL were determined by serial dilution and plating on LB-Miller agar.