Spectral analysis and sorting of microbial organisms using a spectral sorter

This chapter discusses the problems related to the application of conventional flow cytometers to microbiology. To address some of those limitations, the concept of spectral flow cytometry is introduced and the advantages over conventional flow cytometry for bacterial sorting are presented. We demonstrate by using ThermoFisher's Bigfoot spectral sorter where the spectral signatures of different stains for staining bacteria are demonstrated with an example of performing unmixing on spectral datasets. In addition to the Bigfoot's spectral analysis, the special biosafety features of this instrument are discussed. Utilizing these biosafety features, the sorting and patterning at the single cell level is optimized using non-pathogenic bacteria. Finally, the chapter is concluded by presenting a novel, label free, non-destructive, and rapid phenotypic method called Elastic Light Scattering (ELS) technology for identification of the patterned bacterial cells based on their unique colony scatter patterns.