Biofilms were grown on
glass coverslips (13
mm Ø, no. 1.5 thickness) in a rolling biofilm bioreactor system48 (link) (20 rpm) in FAB 10 mM glucose medium, inoculated
with diluted (OD600 nm = 0.01) bacteria from overnight cultures in
LB. For the biofilm samples that were treated with6f , a concentration of 10 μM was supplemented to the bioreactor’
media at the start of the experiments. The biofilms were cultivated
at 30 °C for 24 h, then washed in PBS to remove loosely attached
cells and incubated for a further 6 or 24 h in fresh medium supplemented
with various treatments. These included free ciprofloxacin 60 μg/mL
(× 300 the MIC of planktonic P. aeruginosa cells,44 (link)6f at 20 μM and ciprofloxacin
in combination with6f . Biofilms exposed to each treatment
were washed in PBS, and the viability of attached cells was evaluated
by fluorescent staining using the LIVE/DEAD BacLight bacterial viability
kit (Molecular Probes, Life Technologies) according to manufacturer
instructions. Following staining, coverslips were rinsed with distilled
water and imaged using a LSM700 AxioObserver (Carl Zeiss, Germany)
confocal laser scanning microscope (CLSM). Viable and nonviable biofilm
biomass quantification from image stacks of biofilms was done with
Fiji-ImageJ software. Live/dead ratios were established for each treatment
and compared to untreated controls.
glass coverslips (13
mm Ø, no. 1.5 thickness) in a rolling biofilm bioreactor system48 (link) (20 rpm) in FAB 10 mM glucose medium, inoculated
with diluted (OD600 nm = 0.01) bacteria from overnight cultures in
LB. For the biofilm samples that were treated with
media at the start of the experiments. The biofilms were cultivated
at 30 °C for 24 h, then washed in PBS to remove loosely attached
cells and incubated for a further 6 or 24 h in fresh medium supplemented
with various treatments. These included free ciprofloxacin 60 μg/mL
(× 300 the MIC of planktonic P. aeruginosa cells,44 (link)
in combination with
were washed in PBS, and the viability of attached cells was evaluated
by fluorescent staining using the LIVE/DEAD BacLight bacterial viability
kit (Molecular Probes, Life Technologies) according to manufacturer
instructions. Following staining, coverslips were rinsed with distilled
water and imaged using a LSM700 AxioObserver (Carl Zeiss, Germany)
confocal laser scanning microscope (CLSM). Viable and nonviable biofilm
biomass quantification from image stacks of biofilms was done with
Fiji-ImageJ software. Live/dead ratios were established for each treatment
and compared to untreated controls.
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