Two-dimensional fluorescence fingerprinting using a rapid scanning fluorometer

Abstract

A rapid scanning fluorometer is used for the fluorescence fingerprinting of bacteria and environmental pollutants. The fingerprint data are formatted as a two-dimensional array of fluorescence intensity as a function of multiple emission and excitation wavelengths. These emission-excitation matrix (EEM) data are utilized as fluorescence spectral signatures in the qualitative and quantitative analyses of clinically important bacteria and the class of environmental pollutants known as polynuclear aromatic hydrocarbons (PNAs). The EEM fingerprinting technique is used to differentiate among six strains of Pseudomonas aeruginosa and one strain of Pseudomonas fluorescens based on the selective extraction of diffusable fluorescent pigments, produced by these microorganisms. The EEM fingerprint is also the basis for a response ratio method for detection of non-fluorescent bacteria through selective dye adsorption. The differentiation of Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Enterobacter, Streptococcus and Staphylococcus species is achieved using this procedure. The rapid EEM fingerprinting of PNAs is afforded by the development of instrumentation and methods for automated sample preparation with rapid scanning fluorescence detection. Flow injection analysis (FIA) principles are utilized in the automation of a multiple solvent extraction procedure. The rapid scanning fluorometer, video fluorometer, is also used as a detector for high performance liquid chromatography in the analysis of a very complex environmental sample. Several PNAs are identified using this combination of instrumentation and special methods of data selection and analysis. These techniques can be employed for totally automated, unambiguous fingerprinting of environmental pollutants.