XTT assay for biofilm metabolic activity

Pre-formed biofilms of S. aureus and P. aeruginosa were grown for 24 ​h in different media (as described above) under monospecies and mixed-species conditions. The next day, biofilms were washed once with 150 ​μL of respective media (LB, FBS or IVWM), after removing suspended media. Menadione (SRL, India, 61495) solution (7 ​mg/mL) was diluted 1:100 in sterile distilled water. A mixture of LB:XTT:Menadione in 79:20:1 ratio was freshly prepared [35] (XTT, Invitrogen, USA, X6493), and 150 ​μL of this mixture was added to each well (including media only controls). The plates were covered in aluminum foil and incubated for 4 ​h at 37 ​°C under static conditions. From each well, 100 ​μL was transferred to a new 96-well plate and absorbance was measured at 492 ​nm. For each set of biofilm conditions, metabolic activity was normalized to log₁₀ (CFU) of the biofilm, which was calculated from replicate biofilms that had been set up simultaneously. For the mixed-species biofilms, metabolic activity was normalized to the log₁₀ (total CFU) of both species in the biofilm.