Raman microspectroscopy for species identification and mapping within bacterial biofilms

: A new method of mapping multiple species of oral bacteria in intact biofilms has been developed, using the optical technique of confocal Raman microscopy. A species classification algorithm, developed on dried biofilms, was used to analyze spectra of hydrated biofilms containing two microbial species central to dental health: Streptococcus sanguinis and Streptococcus mutans. The algorithm transferred successfully to the hydrated environment, correctly identifying the species of origin of single-species biofilms. We then used the algorithm successfully both to detect the presence of two species in mixed biofilms and to create spatial maps within these biofilms.