Coupled Turbidity and Spectroscopy Problems: A Simple Algorithm for Volumetric Analysis of Optically Thin or Dilute, In Vitro Bacterial Cultures in Various Media.

An approach binary spectronephelometry (BSN) to perform real-time simultaneous noninvasive in situ physical and chemical analysis of bacterial cultures in fluid media is described. We choose to characterize cultures of Escherichia coli (NC), Pseudomonas aeruginosa (PA), and Shewanella oneidensis (SO) in the specific case of complex media whose Raman spectrum cannot be unambiguously assigned. Nevertheless, organism number density and a measure of the chemical makeup of the fluid medium can be monitored noninvasively, simultaneously, and continuously, despite changing turbidity and medium chemistry. The method involves irradiating a culture in fluid medium in an appropriate vessel (in this case a standard 1 cm cuvette) using a near infrared laser and collecting all the backscattered light from the cuvette, i.e., the Rayleigh-Mie line and the inelastically emitted light which includes unresolved Raman scattered light and fluorescence. Complex "legacy" media contain materials of biological origin whose chemical composition cannot be fully delineated. We independently calibrate this approach to a commonly used reference, optical density at 600 nm (OD600) for characterizing the number density of organisms. We suggest that the total inelastically emitted light could be a measure of the chemical state of a biologically based medium, e.g., lysogeny broth (LB). This approach may be useful in a broad range of basic and applied studies and enterprises that utilize bacterial cultures in any medium or container that permits optical probing in the single scattering limit.

Optical interference probe of biofilm hydrology: label-free characterization of the dynamic hydration behavior of native biofilms.

Biofilm produced by Escherichia coli (E. coli) or Pseudomonas aeruginosa (P. aeruginosa) on quartz or polystyrene is removed from the culture medium and drained. Observed optical interference fringes indicate the presence of a layer of uniform thickness with refractive index different from air-dried biofilm. Fringe wavelengths indicate that layer optical thickness is < 20 ?? ? m or 1 to 2 orders of magnitude thinner than the biofilm as measured by confocal Raman microscopy or fluorescence imaging of the bacteria. Raman shows that films have an alginate-like carbohydrate composition. Fringe amplitudes indicate that the refractive index of the interfering layer is higher than dry alginate. Drying and rehydration nondestructively thins and restores the interfering layer. The strength of the 1451-nm near infrared water absorption varies in unison with thickness. Absorption and layer thickness are proportional for films with different bacteria, substrates, and growth conditions. Formation of the interfering layer is general, possibly depending more on the chemical nature of alginate-like materials than bacterial processes. Films grown during the exponential growth phase produce no observable interference fringes, indicating requirements for layer formation are not met, possibly reflecting bacterial activities at that stage. The interfering layer might provide a protective environment for bacteria when water is scarce.