Swarming Assay for P. aeruginosa

We prepared swarming medium to generate branching patterns of P. aeruginosa according to the following recipe adapted from [21 (link)]: 200 ml of 5x stock phosphate buffer, 1 ml of 1 M MgSO₄, 1 ml of 0.1 M CaCl₂, casamino acids stock solution (200 g/L), agar stock solution (1.25%, melted), and sterilized water to make up 1 liter. The volumes of casamino acids and agar were determined by the required final concentrations. To make 1L 5x phosphate buffer stock solution, we dissolved 12 g Na₂HPO₄ (anhydrous), 15 g KH₂PO₄ (anhydrous), and 2.5 g NaCl in water and sterilized by autoclaving. To make 200 ml casamino acids stock solution, we dissolved 40 g casamino acids (Gibco™ Bacto™ 223120) in water and sterilized by filtering. To make 1L agar stock solution, we added 12.5 g granulated agar (BD Difco™ 214530) in water and sterilized by autoclaving. Each swarming plate was prepared by pipetting exactly 20 ml of medium into a petri dish (100 mm, Falcon), and the dish was allowed to cool for 20 min to 1 hour.