Towards real-time monitoring of bacterial cultures without the need for physical sampling: elastic scattering, fluorescence and Raman spectroscopy of Escherichia coli cultures.

The first part of this dissertation pertains to the fringe analysis of a hydration layer that forms spontaneously when biofilms dry. This layer at optical thickness ˂ 20 µm is approximately 1-2 orders of magnitude thinner than the biofilm thickness as measured by confocal Raman microscopy and confocal fluorescent microscopy. Drying/rehydration experiments performed on films withstood multiple cycles, returning to the same optical thickness proving their robust nature. The strength of a distinct water peak (1451 nm) in addition to others, reflects the thickness of the hydration layer. Confocal Raman microscopy showed that biofilms are chemically very similar to pure alginate (synthetic) films. This shows that spontaneous formation of the hydration layer is a chemical process verses a biological process. The second chapter presents real-time, noninvasive, quantitative analysis of bacterial cultures for cell count and glucose uptake using turbidity corrected spectroscopy and Raman. We hypothesize that a Raman feature we ascribe to “biomass” (~1060 cm-1) i.e. a υ(C-O-C) asymmetric stretch or (C-C) stretch, and a glucose peak (~1140 cm-1) i.e. the υ(C–O) and/or υ(C–C) stretch allow for the real-time monitoring of cell count and nutrient depletion. We calibrated this technique to be backward compatible with OD600 for characterizing culture number density. The method involves irradiating a culture in fluid medium (LB/MM) in a quartz cuvette using a NIR laser (785 nm) and collecting all backscattered light from the cuvette. This approach may be useful in a broad range of academic/industrial applications that utilize bacterial cultures