On December 28, all of the cyclone's convection was removed well east of the circulation center due to moderate to strong westerly vertical wind shear.
Upon completion of the incubation, planktonic cells were removed, wells were washed twice with PBS and 200 μL of SaniDade 12.0 (BioSafe System, Hartford, CT, USA) diluted as per manufacturer's recommendations and were loaded into the wells.
This reagent was removed, wells washed, and tetramethylbenzidine added as substrate.
Subsequently the medium was removed, wells were washed with PBS (phosphate buffered saline), and adherent bacteria were stained with crystal violet (0.2% in ddH2O, room temperature, 10 min).
After 1 hr at 37°C in 5% CO2, culture media supernatant was removed, wells washed twice with warm PBS at 37°C and fresh media was added.
After 22 h incubation, the medium was removed, wells were washed three times with PBS, filled with 1 ml of complete medium and incubated for 1 h at 37°C with 5% CO2.
Supernatants were removed, wells were washed with ELISA wash buffer, and NE protein released was detected with the ELISA kit.
After formazan generation became non-linear (> 10 min), the XTT/PMS was removed, wells were washed twice, and the first 64 wells were used for LDH measurement.
After 48-h incubation, medium was removed; wells were supplemented with 200 μL/well DMSO and incubated in the dark for 15 min with gentle agitation.
After 30 h, medium was removed, wells were gently rinsed with HBSS, cultures were fixed with 5% buffered formalin in DMEM.
