Development of a multispectral light-scatter sensor for bacterial colonies

We report a multispectral elastic-light-scatter instrument that can simultaneously detect three-wavelength scatter patterns and associated optical densities from individual bacterial colonies, overcoming the limits of the single-wavelength predecessor. Absorption measurements on liquid bacterial samples revealed that the spectroscopic information can indeed contribute to sample differentiability. New optical components, including a pellicle beam splitter and an optical cage system, were utilized for robust acquisition of multispectral images. Four different genera and seven shiga toxin producing E. coli serovars were analyzed; the acquired images showed differences in scattering characteristics among the tested organisms. In addition, colony-based spectral optical-density information was also collected. The optical model, which was developed using diffraction theory, correctly predicted wavelength-related differences in scatter patterns, and was matched with the experimental results. Scatter-pattern classification was performed using pseudo-Zernike (GPZ) polynomials/moments by combining the features collected at all three wavelengths and selecting the best features via a random-forest method. The data demonstrate that the selected features provide better classification rates than the same number of features from any single wavelength. Three wavelength-merged scatter pattern from E. coli.