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.