Xtt tetrazolium salt

Subsequent to the appropriate incubation of the microtiter plates, medium was
aspirated from the wells and nonadherent cells were removed by washing the
biofilms as described above. Colorimetric XTT reduction assay of biofilm was
performed as previously reported52 (link)53 (link). 0.5 gm/L stock
solution of XTT tetrazolium salt (Sigma) in PBS was filter sterilized through
0.22 μm pore size filter and stored in aliquots at
−80 °C. Just prior assay, an aliquot was thawed and
1 μM final concentration of freshly prepared menadione (Sigma)
was added to the XTT solution. Hundred (100) μl of XTT-menadione
solution was distributed into the wells containing prewashed biofilms and to the
empty wells (for the background values of XTT reduction) and incubated at
37 °C in the dark for 1 hr. Colorimetric change in the
XTT reduction (reduced formazan-coloured product formation which is correlated
with the metabolic activity of the biofilm) was measured in a microtiter plate
reader at 492 nm.

Biofilms were formed in a 96-well microtiter plate as described previously66 (link)67 (link). For inoculum preparation, exponential YPD broth cultures were harvested, and cells were resuspended in RPMI-1640 medium at a density of 1 × 10⁶ cells/ml. 100 μl of inoculums were dispensed into selected wells of 96-well microtiter plates and incubated at 37 °C for 2 days. After incubation, medium was gently aspirated from the wells and non-adherent cells were removed by washing thrice with sterile PBS. Residual PBS of wells was then removed by blotting with paper towels. Colorimetric XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium- 5-carboxanilide sodium salt] reduction assay was then performed for the quantification of biofilm formation as previously reported66 (link)67 (link). Briefly, 1 μM final concentration of menadione (Sigma; 10 mM prepared in acetone) was added to the filter sterilised (0.22 μm filter) stock solution of XTT tetrazolium salt (0.5 g/L) (Sigma). 100 μl of XTT-menadione solution was added into the prewashed preformed biofilms and to empty wells (for the background values of XTT reduction) of microtiter plates and incubated at 37 °C in the dark for 1 hr. Colorimetric change in the XTT reduction was measured in a microtiter plate reader at 492 nm.