Broth Microdilution Method for CS-NPs MIC

The broth microdilution method was used to determine the MIC of CS-NPs [12] . Two-fold serial dilutions of CS-NPs in Muller-Hinton broth (Oxoid, UK) were prepared to reach a final volume of 0.1 mL of each concentration in each well. A standardized inoculum using the direct colony suspension was prepared and diluted in sterile saline to obtain the suspension's final concentration of 0.5 McFarland. Then the inoculum was diluted at 1:20 to yield 5 × 10⁶ CFU/mL. Finally, 0.01 mL of this suspension was added to each well. Negative control tubes containing broth only and other negative control tubes containing CS-NPs only with different concentrations. The microtiter plate was incubated at 37 °C for 24 hrs.
The MIC of CS-NPs against P. aeruginosa clinical isolates was determined using the resazurin microtiter plate assay[15] (link). This assay uses the redox indicator resazurin (Sigma-Aldrich, Germany) that changed color from blue to pink in the presence of viable cells. The MIC was determined as the concentration at which there was no color change following 4 hrs incubation of the overnight cells with 0.015% resazurin (Figure 1). One dilution below the MIC was regarded as the sub-MIC concentration and was used to evaluate the ability of the CS-NPs to inhibit the virulence activity.