Optical fiber-based synchronous fluorescence spectroscopy for bacterial discrimination directly from colonies on agar plates

Abstract

The development of experimental conditions for rapid bacterial discrimination using fluorescence spectroscopy fingerprinting is presented. Colonies of Pseudomonas and related reference strains on agar plates were analyzed directly using an optic fiber coupled to a laboratory spectrofluorimeter. Spectra were collected using either classic fluorescence spectroscopy after excitation at 250 nm and 340 nm for aromatic amino acids and nucleic acids (AAA + NA) and nicotinamide adenine dinucleotide (NADH) respectively, or synchronous scanning in the excitation wavelength range 250-500 nm. Factorial discriminant analysis (FDA) showed 100% correct classification at the genus and species level from NADH spectra and 100% correct classification at the genus and species level for 6l ¼ 30, 70, 90 and 110 nm (cross-validation). Analysis of variance (ANOVA) confirmed that culture time (48 or 72 h) colony and optic fiber positioning had non-significant impacts on differences between species. The use of optical fiber-fluorescence spectroscopy for bacterial discrimination directly on colonies is fast, simple and reliable. The results are independent of culture growth phase and neither need reagent addition nor prior manual preparation of cells, thus eliminating all risk of human error or contamination during sample work-up.a UPRES 2008.03.0101 Typicit e des Produits alimentaires, VetAgro

Optical Fiber-Based Steady State and Fluorescence Lifetime Spectroscopy for Rapid Identification and Classification of Bacterial Pathogens Directly from Colonies on Agar Plates

“…Steady state fluorescence spectra of the bacterial colonies were recorded using Fluorolog spectrofluorometer (Flurolog, HORIBA instruments Inc., USA) coupled with optical fiber. The optical fiber was placed at 9 mm above the colonies [ 6 ]. The excitation source (450 W Xenon lamp) is coupled to the double excitation monochromator to obtain the light of a desired wavelength.…”

“…Fluorescence lifetime measurements of tryptophan were obtained using Time Correlated Single Photon Counting System (TCSPC, HORIBA instruments Inc., USA) coupled with optical fiber. The optical fiber was placed at 9 mm above the colonies [ 6 ]. The bacterial colonies were excited using 280 nm Nano LED (Pulse width: <1 ns), with a fast response red sensitive PMT (Hamamatsu Photonics, Japan) detector.…”

“…Traditional methods for bacterial identification based on morphology and biochemical tests such as bacterial cultures, DNA-based methods, and antibody-based detection scheme are laborious and reagents- and time-intensive [ 3 ]. Fast methods such as flow cytometry, direct epifluorescent filter technique (DEFT), ATP-based bioluminescence, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) [ 4 , 5 ], and impedancemetry were developed for identification and classification of bacteria [ 6 ]. However, these methods are also reagent- and time-intensive.…”

“…For all these studies, fluorescence spectra were acquired for bacteria grown on a liquid medium and suspended in saline solution after centrifugation and washing. Recently, Belal et al [ 6 ] reported the use of optical fiber-based synchronous fluorescence spectroscopy to discriminate colonies of Pseudomonas and related reference strains directly on agar plate. The authors also used classic fluorescence at 250 nm and 340 nm excitation for aromatic amino acids and nucleic acids (AAA + NA) and nicotinamide adenine dinucleotide (NADH), respectively.…”

Identification of bacteria by poly-aromatic hydrocarbons biosensors

“…Another leading approach for bacterial identification is the utilization of fluorescence spectroscopy. [34][35][36][37][38][39][40][41][42][43][44][45][46] Microorganisms are consist of several components such as the amino acids Tyrosine, Tryptophane, and Phenylalanine, or metabolites such as NADPH that can be detected based on their intrinsic fluorescence. [47,48] DNA also fluoresce under UV-VIS irradiation, however, its native emission is usually too low to be detected.…”