HOLOGRAPHIC CHARACTERIZATION OF MICROBES

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Long-term Objectives To describe generally useful real-time light-scattering methods for detecting, identifying and counting motile microbes. To describe methods of obtaining species specific motion spectral "signatures" of microbes in aqueous media. To demonstrate research equipment which can identify individual microbes in situ in natural mixtures, against various backgrounds such as detrital microparticulate and organic macromolecules. To begin to build a "library" of the motion spectral signatures and dynamic holograms associated with motile microbes which impact our society or our environment, e.g., Salmonella, E. coli. To show how these techniques complement flow cytometry and other methods currently used to sort and study unicellular microbes. To point the way to using similar non-intrusive techniques in a wide variety of situations to improve human health and wellbeing. Experimental Design Prototype apparatus has been built from a commercially available Laser Doppler Velocimeter. The Doppler shifts of coherent light scattered from the moving parts of motile microbes can be detected by optical heterodyning techniques. Ensemble averaging of the resulting spectra enhances signal to noise ratios and gives "motion spectra" characteristic of the microbes. Simultaneously, dynamic holograms form around the axis of the laser beats exiting the sample. These holograms contain all the information require to optically characterize the sample. For example, it is theoretically possible to store a set of dynamic holograms in an optically active crystalline medium by rotating the crystal during reception of the time-series information. If the scattered light from a second similar suspension of microbes was used as input to this crystal store, the reference beam would reconstruct (and be readily detectable) during the rotation of the crystal, during passage through an arc corresponding to that used for storage of the original signature. Holographic and spectroscopic techniques are completely complementary. The former promises fast optical processing of signatures and cell count from samples containing many microbes, while the latter promises to allow continuous quantitative analysis of mixtures of microbes and particulate at very low concentration in drinking water, wastewater and groundwater.