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

Abstract

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.

Noninvasive Blood and Tissue Analysis: Raman Spectroscopy, One Perspective for Monitoring of Glucose and Beyond

“…Current efforts are now focused on including glucose in the PVOH platform. Table 1 includes the vital signs/variables for which there is preliminary published data [38][39][40][41][42][43] that could result from a new PVOH configuration along with technical comments of feasibility and differences from currently used technologies. Elastic scattering and fluorescence/Raman activity of a tissue, turbid medium, object, or material can be used for imaging fingertips 10 or spinal cords 42 and other applications with a PVOH.…”

“…Vital Signs/Variables for Which There Is Preliminary Published Data[38][39][40][41][42][43] That Could Result From a New PVOH Configuration Along With Technical Comments of Feasibility and Differences From Currently Used Technologies.…”

In vivo, noncontact, real-time, PV[O]H imaging of the immediate local physiological response to spinal cord injury in a rat model

“…To date, we have validated PV[O]H in human and rat model studies involving skin, bacterial cultures in various media, and in unambiguous inanimate model systems. [10][11][12][13] The meaning/nature of the quantities calculated using PV[O]H depends critically on context. When applied to skin that has a capillary vascularization, PV[O]H calculates changes in the hematocrit (Hct) and total vascular volume of the capillary network.…”

PVOH and spontaneous Raman-based images of spinal cord in the immediate aftermath of localized contusive injury obtained noninvasively and in vivo in a rat model using remitted light from a single spatially scanned laser

“…We employed the PVOH algorithm to provide contrast for imaging applications based on turbidity expecting that 1) physical debris from the injury itself could provide increased turbidity and 2) perhaps spillage of cell contents could trigger the immune system and then cascading coalescence growth chemistry. Elastic scattering occurs whenever light traverses any medium containing inhomogeneities in the refractive index, and in a background of plasma one could expect to detect platelets, bacteria 5 , and possibly viral loads. Cerebrospinal fluid contains much less protein than plasma and should provide an even more favorable background against which other spectral and propagation changes might be observed and quantified.…”