Resazurin Microtiter Assay (REMA) Plates

We used resazurin as color indicator to measure the MIC of S. pneumoniae R6wt and R6ΔPezAT mutant strains. Resazurin is a blue non-fluorescent dye that can be converted to the pink fluorescent dye, resorufin, by metabolically active cells (Tizzard et al., 2006; Mania et al., 2010; Khalifa et al., 2013), a method that has been used for S. pneumoniae (Patel et al., 2011; Vandevelde et al., 2014). The color changes can be also measured by light absorbance at 570 nm for resorufin or 600 nm for resazurin. MICs of various antibiotics (ampicillin, benzetacil, levofloxacin, and streptomycin) were determined by using twofold serial dilution method. Briefly, the cells were grown at 37°C until OD₆₅₀∼0.3. Cells were diluted to 1:100 into pre-warmed fresh medium and 100 μl were added into a flat-bottom BD Falcon 96-Well Cell Culture Plate. Different amounts of antibiotics and 200 μM resazurin were added to each well. The mixtures were incubated at 37°C and the color changes were measured at 600 nm every hour by employment of a Varioskan Flash Multimode Reader (Thermo Scientific). These assays were repeated at three times and the mean values and the standard deviations were calculated. t-test (with p-value < 0.05) was used to evaluate the significant differences of the results. The results were validated by the use of standard MIC determinations on microtiter plates (Andrews, 2001), without resazurin but determining the number of colonies formed by cultures grown in the absence or in the presence of ampicillin.