Polystyrene microtiter 96 well plates

For this purpose, an overnight culture of P. mirabilis isolates was diluted (1 : 100) in TSB to reach the 0.5 McFarland concentration, and 5 μl of the diluted culture was inoculated in each well of polystyrene microtiter 96-well plates (Greiner, Germany). After 24 h incubation of the plates at 37°C, the medium was removed, and the wells were washed carefully with double distilled water (DDW) and fixed by adding 200 μl of ethanol (96%) for 15 minutes. Next, the medium was removed, and the plate was left to dry. The biofilms were stained with 0.1% (w/v) crystal violet for 20 minutes at 37°C. Next, the biofilm was washed three times with DDW. Then, 200 μl of ethanol–acetic acid (90 : 10) was added to each well, and optical density (OD) was measured at 590 nm with an enzyme-linked immunosorbent assay (ELISA) microtiter plate reader. Each assay was done in triplicate, and the mean absorbance ± standard deviation was calculated for all repetitions of the tests. Escherichia coli K12 and Pseudomonas aeruginosa ATCC 27853 were used as negative (weakly biofilm-forming) and positive (strong biofilm-forming) control strains, respectively [19 ].