Crystal violet

P. aeruginosa isolates were seeded on LB agar (Neogen, Lansing, MI, USA) and incubated at 37 °C for 24 h. One colony of each isolate was inoculated in the presence (1/2, 1/4, and 1/8 MIC) or absence (control) of subinhibitory concentrations of bio-AgNPs at the bottom of twitching agar plates containing 1.0% w/v tryptone (Acumedia, Lansing, MI, USA), 0.5% w/v yeast extract (BD, Sparks, MD, USA), 1.0% w/v sodium chloride (Merck, Darmstadt, Germany), and 1.0% w/v agar (BD, Sparks, MD, USA). Plates were inverted and incubated at 37 °C for 24 h. Posteriorly, the agar was removed and stained with 2% w/v crystal violet (Laborclin, Pinhais, PR, Brazil) for 2 h [24 (link)]. The motility halo was measured to the nearest millimeter. As a negative control, each isolate was inoculated in tryptone soy agar (BD, Sparks, MD, USA) under the same conditions.

P. aeruginosa isolates were seeded on LB agar (Neogen, USA) and incubated at 37°C for 24 h. Then, one colony of each isolate was inoculated, in the presence and absence (control) of ½ MIC bio-AgNPs, to the bottom of twitching agar plates containing 1.0% tryptone (Acumedia, USA), 0.5% yeast extract (Bacto, Difco, USA), 1.0% sodium chloride (Synth, Brazil), and 1.0% agar (Acumedia, USA). Plates were inverted and incubated at 37°C for 24 h. Subsequently, the agar was carefully removed, and the motility zone was measured to the nearest millimeter after staining with 2% crystal violet (Laborclin, Brazil) for 2 h (Otton et al., 2017 (link)). As a negative control, each isolate was inoculated in tryptone soy agar (Difco, USA) under the same conditions.

Biofilms were formed as described by Kudirkiene et al. (2012 (link)), with modifications. Briefly, 200 μl of the bacterial suspension containing 10⁴ cells prepared in MH broth and MH with 5% of chicken juice was added in 96-well plates. For biomass formation, the plates were incubated for 48 h at 37°C in microaerophilic conditions.
After incubation, the media were removed, the wells were washed twice with 0.9% NaCl solution and dried for 30 min at 55°C. Total biomass was measured by fixation with 0.1% Crystal Violet (LaborClin) for 5 min, followed by elution with alcohol-acetone solution, containing 80% of ethanol and 20% of acetone (Synth®). The eluted dye was removed from each well and placed in a new 96-well microtiter plate for reading at OD₅₉₅ (BA–bacteria adhered). The assays were done with eight replicates for each strain in three replicates. For the determination of the Biofilm Formation Index, the following formula was used:
$\text{BFI}=\frac{\text{BA}-\text{PC}}{\text{BS}}$
Where BFI represents the final result regarding the Biofilm Formation Index, BA the optical density obtained in the mixture of bacteria adhered, PC the absorbance value in the control wells without microorganisms, BS the optical density (OD₆₀₀) of the suspended cultures in MH and MH with 5% of chicken juice (Naves et al., 2008 (link)). The final classification followed Table 1.