In biofilms, diffusion may limit the chemical activity of nutrients, toxic compounds, and medicines. This study provides direct, noninvasive insight into the factors that will most effectively limit the transport of antibiotics and biocides in biofilms. Self-diffusion coefficients have been determined for a number of fluorescent probes in biofilms of Streptococcus mutans using fluorescence correlation spectroscopy. The effects of probe size and charge and the roles of biofilm pH, ionic strength, and heterogeneity were studied systematically. The relative diffusion coefficients (D in the biofilm divided by that in water) decreased with increasing probe size (3,000-molecular-weight [3K], 10K, 40K, 70K, and 2,000K dextrans). Studies using variably charged substrates (tetramethylrhodamine, Oregon Green, rhodamine B, and rhodamine 6G) showed that the self-diffusion coefficients decreased with an increasing negative charge of the fluorescent probes. No significant effect was observed for changes to the ionic strength (10 ؊4 to 10 ؊1 M) or pH (4 to 9) of the biofilm. Biofilm heterogeneity was responsible for variations of ca. one order of magnitude in the diffusion coefficients.Biofilms are complex suprastructures in which bacterial microcolonies are dispersed in a matrix of extracellular polymeric substances (EPS; polysaccharides, proteins, and DNA), lipids, and other metabolites (3,6,11,14,21). Due to the functional groups on the EPS (e.g., carboxylate, pyruvate, sulfate, etc.), the biofilm generally has an overall negative charge and a high water content (21). The specific structure of biofilms is thought to provide them with a high level of resistance to antibiotics, disinfectants, and detergents (7,19,20). For example, it has been reported that the minimum antibiotic concentration to kill bacteria found in biofilms was about 100 to 1,000 times greater than what was observed for the planktonic organisms (19). A long-standing explanation for the observed increased tolerance to antibiotics is that the biofilm constitutes an effective barrier to the penetration of antimicrobial agents, which is related to a reduction in their diffusive flux with respect to that observed in water (18,19). To that end, it is vital to quantify diffusion in biofilms (23), preferably with noninvasive methods, such as fluorescence correlation spectroscopy (FCS) (4, 15) or attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) (17). Unfortunately, no consensus yet exists on the role of biofilms in diffusion. Relative diffusion coefficients (defined as the diffusion coefficient, D, in a biofilm divided by that in water) vary greatly across the literature.In this paper, the effects of substrate size and charge and of biofilm pH and ionic strength were studied systematically for a biofilm of Streptococcus mutans. S. mutans is a main constituent of dental plaque, which can form dense biofilms both in vivo and in vitro (16). The diffusion coefficients of particle-size standards (dextrans of 3,000 molecular weight [3K], 10K...