The distribution of a carotene-containing yeast (CCY) in a biofilm formed by a small colony variant (SCV) of <i>Pseudomonas aeruginosa</i> PA01 was followed by confocal Raman microspectroscopy (CRM). SCV PA01 and CCY cells were distinguished by their spectral signatures, and the distribution of the overall biomass was monitored by the C–H bending or stretching signal. The distributions of total biomass, PA01, and CCY cells were compared at various times and positions within the biofilm. The distribution of the CCY was very heterogeneous. It was found in the water channels as well as in regions within biofilm colonies. Many of the yeast cells observed within the biofilm colonies under conditions of low or stopped flow were removed when medium was flowing, suggesting that the yeast was not held in the matrix as tightly as were the bacteria.

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