M9 salts medium supplemented with 0.5% glucose was used as the minimal medium. The swarm medium contained 10 g tryptone, 10 g NaCl, and 5 g of glucose L−1 the final agar concentration was 0.5%. The swim medium contained the same constituents solidified with 0.3% agar. To better visualize swarming colonies, a vital dye, triphenyl tetrazolium chloride (TTC), was added to achieve a final concentration of 0.05% when required. Both swim and swarm plates were allowed to dry overnight at room temperature before use. Antibiotics were added, when appropriate,

at the following concentrations: kanamycin at 100 μg mL−1 and rifampicin at 100 μg mL−1. To observe swarming motility, 1 μL culture incubated for 10 h in lysogeny broth (LB) (adjusted to 0.5 OD600 nm) was inoculated onto a thin layer of solid swarm media in a Petri dish (6 mL media per plate). The plates were directly observed at × 400 magnification learn more under an Olympus inverted microscope IX71 in a room heated to 30 °C. Sterile slides were occasionally used instead of Petri dishes to achieve better visualization. The slides were PLX4032 concentration submerged in swarm media, which was solidified with

0.5% agar, to obtain a thin layer of media on the surface and dried at 37 °C briefly before use. After inoculation, the bacteria on the surface of the media were observed under the inverted microscope. Images were recorded using a video camera. For negative staining, formvar-coated TEM grids (copper, 75 mesh) were floated on a drop of bacterial cells suspended in phosphate-buffered saline (PBS, pH 7.4) for 5 min to allow the adhesion of bacterial cells. The isothipendyl grids were stained for 5 min using 2% phosphotungstate. After staining, these

were rinsed with water and then air dried. For ultrathin sectioning, bacteria were washed and suspended in PBS, fixed in 0.2% v/v glutaraldehyde, and embedded in Spurr resin. The specimens were examined with a transmission electron microscope (Philips Tecnai 10). Mutagenesis was performed according to the method described by de Lorenzo et al. (1990). Citrobacter freundii and E. coli S17-1 (λpir)/pUT mini-Tn5-Km were grown overnight in LB media with rifampicin and kanamycin, respectively. A 100-μL aliquot of each culture was mixed in 5 mL of 10 mM MgSO4 and filtered through a 0.45-μm cellulose membrane filter. The filter was then placed on the surface of an LB plate and incubated at 37 °C for 10 h. The bacteria on the filter surface were washed and suspended in 2 mL of 10 mM MgSO4. About 100 μL of the resulting bacterial suspensions was spread onto LB plates containing kanamycin and rifampicin and incubated at 37 °C for ∼36 h. The antibiotic-resistant bacteria were then transferred to swarm agar plates and incubated at 37 °C for 12 h. All swarming-defective colonies were selected.