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We mapped the space–time distribution of stationary and swarmer cells within a growing Proteus mirabiliscolony by infrared (IR) microspectroscopy. Colony mapping was performed at different positions between the inoculum and the periphery with a discrete microscope-mounted IR sensor, while continuous monitoring at a fixed location over time used an optical fiber based IR–attenuated total reflection (ATR) sensor, or “optrode.” Phenotypes within a single P. mirabilispopulation relied on identification of functional determinants (producing unique spectral signals) that reflect differences in macromolecular composition associated with cell differentiation. Inner swarm colony domains are spectrally homogeneous, having patterns similar to those produced by the inoculum. Outer domains composed of active swarmer cells exhibit spectra distinguishable at multiple wavelengths dominated by polysaccharides. Our real-time observations agree with and extend earlier reports indicating that motile swarmer cells are restricted to a narrow (approximately 3 mm) annulus at the colony edge. This study thus validates the use of an IR optrode for real-time and noninvasive monitoring of biofilms and other bacterial surface populations.