Date of Award

Spring 5-15-2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Advisor(s)

Chaiken, Joseph

Keywords

Bacteria, Binary Spectronephelometry, Near Infrared, Principal Component Analysis, Raman, Spectroscopy

Subject Categories

Chemistry | Optics | Physical Chemistry | Physical Sciences and Mathematics | Physics

Abstract

Current noninvasive methods cannot continuously and simultaneously monitor the concentrations of cells and media components that define the state of native bacterial cultures, because of changing turbidity. A new technique, binary spectronephelometry (BSN) has the same or better sensitivity and precision for population monitoring as optical density at 600nm (OD600), while simultaneously measuring metabolic processes. The BSN algorithm uses laser induced emission to probe mildly turbid media i.e., propagation of light occurs in the single scattering regime. A BSN "training set" associates a grid of elastic emission measurements, comprising Rayleigh and Mie scattering, and inelastic emission measurements, comprising fluorescence and Raman emission, with a grid of varying known concentrations of bacteria and media. For the normal lifecycle culture and for some but not all growth media, the results confirmed the linear dependence of the two measured signals on the concentration of bacteria and medium dilutions, and agreement with OD600. Having thus confirmed basic assumptions and a calibrated algorithm, various bacteria cultures were monitored over time, producing quantitative "growth curves" for the bacteria and "depletion curves" for media. Simultaneously obtained Raman spectra demonstrate glucose uptake and conversion into bacterial membrane phospholipids. Beyond the normal life cycle, a few examples of using BSN to monitor cultures subjected to external perturbations are shown. Specific characteristics of Principal Component Analysis (PCA) of Raman spectra, and their variation in bacterial cultures, as well as assessing the role of bacterial transport e.g., diffusion in performing and analyzing BSN, are presented.

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Open Access

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