FIGURE 4.

Quantification of Candida tropicalis NCPF 3111 biofilms after treatment with sessile minimum inhibitory concentration 80 (SMIC80) of Amphotericin B (AmB). (A) Biofilm biomass units (BBU) of biofilms 1 (B1) formed from planktonic culture cells and exposed to AmB (200 μg mL^–1) and biofilms 2 (B2) formed from persister cells (PCs1) that survived the B1 treatment, and were exposed to a second AmB treatment (200 μg mL^–1). (B) Cellular stress metabolites: extracellular and intracellular reactive oxygen species (eROS and iROS) and reactive nitrogen species (RNS) with respect to untreated biofilms. Oxidative stress responses (OSR): superoxide dismutase (SOD), total reduced glutathione (tGSH) and total antioxidant system determined by ferrous reduction antioxidant power (FRAP) assay with respect to untreated condition for B1 and B2 of C. tropicalis. (C) Confocal scanning laser microscopy (CLSM) images of untreated B1 and B2 and after treatment with AmB at SMIC80. Blue channel shows Calcofluor-White in sessile cell walls, and green channel shows the oxidation of the 2′,7′dichlorodihydrofluorescein diacetate probe (DCFH-DA) as an indicator of ROS production inside the biofilms. iROS was quantified as the 2′,7′dichlorofluorescein (DCF) fluorescence by using NIH-Image J. CLSM images showing two-color (blue and green) merge are shown, with a magnification of 600× and scale bar of 5 μm. All experiments were performed in triplicate, for three independent experiments, and the numerical data are presented as means ± standard deviation. *Denotes statistical significance at p < 0.01 for differences when compared with untreated biofilms. ^#p < 0.01 differences considered significant for comparisons between B1 and B2.