Extracellular matrix provides an architectural structure and mechanical stability for aerobic granules. Distributions of cells and extracellular polymeric substances (EPS), including proteins, alpha-and beta-D-glucopyranose polysaccharides, in acetate-fed granules and phenol-fed granules were probed using a novel quadruple staining scheme. In acetate-fed granules, protein and alpha-beta-glucopyranose polysaccharides formed the core, whereas, the cells and beta-D-glucopyranose polysaccharides accumulated in the granule outer layers. Based on these experimental findings, this study indicated that different conclusions can be obtained regarding EPS distributions when granules were stained differently. The core of phenol-fed granules, conversely, was formed principally by proteins; whereas, the cells and alpha- and beta-D-glucopyranose polysaccharides were accumulated at an outer filamentous layer. Using a series of confocal laser scanning microscope (CLSM) images whose threshold values were determined via Otsu's scheme, the three-dimensional distributions of cells and EPS were produced using a polygonal surface model. Structural information extracted can be applied in further development of comprehensive granule models.