Streptococcus pneumoniae forms biofilms, but little is known about its extracellular polymeric substances (EPS) or the kinetics of biofilm formation. A system was developed to enable the simultaneous measurement of cells and the EPS of biofilm-associated S. pneumoniae in situ over time. A biofilm reactor containing germanium coupons was interfaced to an attenuated total reflectance (ATR) germanium cell of a Fourier transform infrared (FTIR) laser spectrometer. Biofilm-associated cells were recovered from the coupons and quantified by total and viable cell count methods. ATR-FTIR spectroscopy of biofilms formed on the germanium internal reflection element (IRE) of the ATR cell provided a continuous spectrum of biofilm protein and polysaccharide (a measure of the EPS). Staining of the biofilms on the IRE surface with specific fluorescent probes provided confirmatory evidence for the biofilm structure and the presence of biofilm polysaccharides. Biofilm protein and polysaccharides were detected within hours after inoculation and continued to increase for the next 141 h. The polysaccharide band increased at a substantially higher rate than did the protein band, demonstrating increasing coverage of the IRE surface with biofilm polysaccharides. The biofilm total cell counts on germanium coupons stabilized after 21 h, at approximately 10 5 cells per cm 2 , while viable counts decreased as the biofilm aged. This system is unique in its ability to detect and quantify biofilm-associated cells and EPS of S. pneumoniae over time by using multiple, corroborative techniques. This approach could prove useful for the study of biofilm processes of this or other microorganisms of clinical or industrial relevance.Microbial biofilms are comprised of both cells and extracellular polymeric substances (EPS) (7).Though a variety of in vitro model systems have been developed for growing and quantifying biofilms, most rely upon the measurement of cells recovered from the surface, using viable plating or direct microscopic counting methods, to estimate the rate or extent of biofilm formation. Even though the EPS may comprise up to 98% of the volume of the biofilm (10), this material is rarely measured. Sutherland (25) noted that cells in pure culture biofilms may produce multiple polysaccharides, but the minute quantities present preclude accurate measurement by wet chemical methods. Streptococcus pneumoniae is suspected of forming biofilms in individuals who develop otitis media (6). S. pneumoniae produces a copious polysaccharide capsule that is a known virulence factor involved in adhesion (12) and that is antiphagocytic, but there have been few published studies documenting biofilm formation by this organism.Real-time monitoring of the biofilms formed when bacteria are in contact with an internal reflection element (IRE) substrate has been performed by Fourier transform infrared (FTIR) spectroscopy. Since biofilms have a different chemical composition than the same organisms in suspension (11,14,16), FTIR spectra show different bands ...