TQ was tested for its potential to prevent biofilm formation of four reference strains (Table 2 ). The TQ was added to the growth medium at the time of inoculation and the cells were allowed to form biofilms [6 (link)]. Prevention of biofilm formation by TQ was examined by microdilution, similar to the MIC assay for planktonic cells. A two-fold serial dilution was prepared in 96-well polystyrene tissue culture plates containing TSB broth with 2% glucose (w/v), with final concentrations of TQ ranging from 0 to 512 μg/ml.
The medium without TQ was used as the non-treated well and the medium with TQ as the blank control. Aliquots of bacterial suspension (10 μl) were inoculated in tissue culture plate wells (5.104 cfu/ml, final concentration). Following incubation at 37°C for 24h, culture supernatants from each well were decanted and planktonic cells were removed by washing three times with phosphate-buffered saline (7 mM Na2HPO4, 3 mM NaH2PO4 and 130 mM NaCl at pH 7.4). Cells in biofilm were fixed with methanol during 15 min, air dried and stained with 1% crystal violet [27 (link)]. Biofilm formation was quantified by measuring the absorbance at 595 nm using a microplate reader (GIO. DE VITA E C, Italy).
In order to asses the ability of TQ to prevent biofilm formation, the percentage of biofilm inhibition was calculated using the equation [(OD growth control _ OD sample)/OD growth control] × 100 [6 (link)]. Each assay was repeated three times.
The minimum biofilm inhibition concentration (MBIC50) was defined as the lowest concentration of TQ that showed 50% inhibition on the biofilm formation.
The medium without TQ was used as the non-treated well and the medium with TQ as the blank control. Aliquots of bacterial suspension (10 μl) were inoculated in tissue culture plate wells (5.104 cfu/ml, final concentration). Following incubation at 37°C for 24h, culture supernatants from each well were decanted and planktonic cells were removed by washing three times with phosphate-buffered saline (7 mM Na2HPO4, 3 mM NaH2PO4 and 130 mM NaCl at pH 7.4). Cells in biofilm were fixed with methanol during 15 min, air dried and stained with 1% crystal violet [27 (link)]. Biofilm formation was quantified by measuring the absorbance at 595 nm using a microplate reader (GIO. DE VITA E C, Italy).
In order to asses the ability of TQ to prevent biofilm formation, the percentage of biofilm inhibition was calculated using the equation [(OD growth control _ OD sample)/OD growth control] × 100 [6 (link)]. Each assay was repeated three times.
The minimum biofilm inhibition concentration (MBIC50) was defined as the lowest concentration of TQ that showed 50% inhibition on the biofilm formation.
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