Biofilms were analyzed at 43 h via confocal microscopy. All procedures
for biofilm formation and topical treatments were as described above,
except that 13.4 μL of 1 mM Alexa Fluor 647 fluorophore-labeled
dextrans (647/668 nm; Molecular Probes, Carlsbad, CA, EUA) was added
to the culture medium to label the extracellular matrix (at 0, 19,
and 27 h). At 43 h, the biofilms were dip-washed on a 24-well plate
containing 0.89% NaCl and incubated in another 24-well plate containing
0.89% NaCl with 1.5 μL of SYTO9 (485/498 nm; Molecular Probes)
(30 min) to label microorganisms.5 (link),40 (link) After, they
were dip-washed on a new 24-well plate with 0.89% NaCl and imaged.
A confocal microscope (Carl Zeiss LSM 800 with Airyscan and a GaAsp
detector, Germany) was used with an EC Plan-Neofluar 20×/0.50
Oil DIC M27 objective, with laser wavelengths (488 nm, 2.10%; and
561 nm, 1.81%), with increments of 1.5 μm. The images were analyzed
using ZEN Blue software to quantify the biomass, maximum thickness,
and percentage of coverage area using COMSTAT2.
Three experimental
occasions were performed. Two discs represented
each treatment group (formulation), and three images were acquired
per disc avoiding the disc’s edges (n = 6).
All data files were used for the quantification analyses, and a representative
image from each group was selected to illustrate the findings.
for biofilm formation and topical treatments were as described above,
except that 13.4 μL of 1 mM Alexa Fluor 647 fluorophore-labeled
dextrans (647/668 nm; Molecular Probes, Carlsbad, CA, EUA) was added
to the culture medium to label the extracellular matrix (at 0, 19,
and 27 h). At 43 h, the biofilms were dip-washed on a 24-well plate
containing 0.89% NaCl and incubated in another 24-well plate containing
0.89% NaCl with 1.5 μL of SYTO9 (485/498 nm; Molecular Probes)
(30 min) to label microorganisms.5 (link),40 (link) After, they
were dip-washed on a new 24-well plate with 0.89% NaCl and imaged.
A confocal microscope (Carl Zeiss LSM 800 with Airyscan and a GaAsp
detector, Germany) was used with an EC Plan-Neofluar 20×/0.50
Oil DIC M27 objective, with laser wavelengths (488 nm, 2.10%; and
561 nm, 1.81%), with increments of 1.5 μm. The images were analyzed
using ZEN Blue software to quantify the biomass, maximum thickness,
and percentage of coverage area using COMSTAT2.
Three experimental
occasions were performed. Two discs represented
each treatment group (formulation), and three images were acquired
per disc avoiding the disc’s edges (n = 6).
All data files were used for the quantification analyses, and a representative
image from each group was selected to illustrate the findings.
