*2.5. Biofilm Study Using XTT Assay*

Biofilms of each isolate were grown for 48 h in separate wells in 96-well plates. Planktonic cells were then removed by washing three times with PBS. The biofilm-containing wells were treated with CSA-131 (100 μg/mL) and incubated for another 24 h. After another PBS wash, 100 μL of a mixed solution of 0.5 mg/mL 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) and 10 mM menadione in acetone was added to each well. Plates were covered in aluminum foil and incubated for 2 to 3 h at 37 ◦C. Remaining solution was removed from each well and remaining dye in the wells was then quantified with a microtiter plate reader at 490 nm. Optical density results of test wells were compared with controls to determine the percent of biofilm remaining in each [23].
