MONITORING MICROBIAL ADHESION AND BIOFILM FORMATION BY ATTENUATED TOTAL REFLECTION FOURIER-TRANSFORM INFRARED-SPECTROSCOPY

A major problem in accurately defining bacterial adhesion mechanisms and processes occurring in biofilms on surfaces is the lack of techniques that nondestructively provide on-line information about the microorganisms, their extracellular polymers, and metabolites. The attenuated total reflectance (ATR) technique of Fourier transform infrared spectroscopy (FT-IR) is ideally suited to monitor molecular interactions at the solution/internal reflection element (IRE) interface, and we report its application to biofilm research. Two methodologies were utilized to obtain the ATR/FT-IR spectra of living Caulobacter crescentus cells attached to germanium crystals. Initially, spectra of attached bacteria in high purity water produced molecular details of the attachment process without spectral interferences from components of the medium. A growth medium, utilized in the second method, allowed direct examination of the infrared absorption bands associated with the actively growing microorganisms on the surface of the IRE in the spectral region of 2000 to 1200 cm-1. Using the amide 11 band as a marker for biofilm biomass, the detection limit was determined to be approximately 5 x 10(5) cells.cm-2. These results proved that the ATR-FT/IR methodologies can be utilized to provide chemical information from bacteria and bacterial products located within approximately 1 mum of the surface without spectral interferences due to components of the medium.