IMPROVEMENTS IN FLOW CYTOMETRY OF BACTERIA & VIRUSES

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Several approaches to increasing the sensitivity of flow cytometry for detection and characterization of small bacteria and large virus particles will be explored, including a water-immersed flow chamber, a laser Doppler light scatter detector system, front end electronics incorporating source noise compensation, and light noise reduction by acousto-optic modulation. A high-sensitivity flow cytometer developed as a result of these studies will be used for measurements of light scattering and fluorescence form bacteria, viruses, and test particles smaller than 0.5 mum in diameter. Two-parameter measurements of light scattering and cyanine dye fluorescence on multiple aliquots of a sample will be used to estimate bacterial membrane potentials and to rapidly determine which of several energy sources would support the growth of an organism and which of several inhibitors would prevent growth. Ratiometric measurements of the fluorescence of the fluorescein derivative BCECF will be used to detect pH gradients between bacterial cytoplasm and the medium. Multistation, multiparameter measurements of light scattering and of the fluorescence of mixtures of dyes, including membrane potential and pH probes, fluorescent stains for protein, DNA and RNA, and fluorogenic enzyme substrates, will be investigated to determine which parameters add the most additional information relevant to bacterial identification.