Multicolor fluorescence experiments are conducted to investigate the distributions of extracellular polymeric substances and/or cells in the bioaggregates. Successful staining requires that the dyes could fully stain the targeted substances of bioaggregates in a finite time. The mass transfer limit for one of the four fluorescent dyes, calcofluor white, concanavalin A conjugated with tetramethylrhodamine, Nile red, and SYTO 63, penetrating entire phenol-fed granules or those sectioned at 50 mu m thick, was quantitatively determined. The former three dyes sufficiently stained the entire granule within prescribed time intervals. However, the SYTO 63 could not penetrate the 600-mu m granule in a finite time. Simplified one-dimensional diffusional model estimated the apparent diffusivity of SYTO 63 in the aerobic granule matrix. This work revealed that each staining scheme should be examined for the possible mass transfer limit of dyes during staining.