Rapid Identification of Microorganisms by Intrinsic Fluorescence (original) (raw)
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Optics …, 2007
Remotely sensed laser-induced autofluorescence spectra of pure cultures of fungal strains (Aureobasidium pullulans, Verticillium sp.) and of bacterial strains (Bacillus sp., Pseudomonas sp.) are presented. The strains were isolated from samples collected in a Roman archaeological site (Tropaeum Traiani) near Constanta, Romania. The fluorescence spectra were detected in vivo from a distance of 25 m in the outdoor, using a high spectral resolution fluorescence LIDAR featuring a UV laser (XeCl@308 nm) as an excitation source. All the examined strains, except for the A. pullulans, showed fluorescence features such to allow their characterisation by processing data with multivariate techniques. Both Principal Component Analysis and Cluster Analysis were applied to the data set and compared to discriminate between the examined strains.
Journal of Chemometrics, 2011
Fluorescence analysis, in particular two-dimensional excitation-emission matrix (EEM) spectroscopy, is a sensitive in-line process monitoring tool in the biotechnological production. Before it can be widely adopted by the industry, fast effective algorithms for the analysis of massive and complex fluorescence data should be developed. Weak emission signals are prone to various interferences complicating the modeling, such as excitation light Rayleigh scatter. The scatter is usually considered as an unwanted background to be avoided or removed.
Rapid Detection of Three Common Bacteria Based on Fluorescence Spectroscopy
Sensors, 2022
As an important part of environmental water quality monitoring, efficient bacterial detection has attracted widespread attention. Among them, LIF (laser-induced fluorescence) technology has the characteristics of high efficiency and sensitivity for bacterial detection. To simplify the experimental process of bacterial detection, fluorescence emission spectra of E. coli (Escherichia coli) and its deactivated controls, K. pneumoniae (Klebsiella pneumoniae) and S. aureus (Staphylococcus aureus), were analyzed with fluorescence excitation by a 266 nm laser. By analyzing the results, it was found that the dominant fluorescence peaks of bacterial solutions at 335~350 nm were contributed by tryptophan, and the subfluorescence peaks at 515.9 nm were contributed by flavin; besides, K. pneumoniae and S. aureus had their own fluoresces characteristics, such as tyrosine contributing to sub-fluorescence peaks at 300 nm. The three species of bacteria can be differentiated with whole fluorescence ...
Applied Spectroscopy, 1985
Time-resolved fluorescence spectra have been obtained for Escherichia coli, Pseudomonas fluorescens, Staphylococcus epidermidis, and Enterobacter cloacae. Pseudomonas fluorescens has been shown to have distinctly different time-resolved spectra. Fluorescence excitation spectra for the three other organisms showing similar time-resolved spectra are very nearly alike, while spectra of Pseudomonas fluorescens are markedly different. This suggests that the changes in the average fluorescence lifetime with emission wavelength are due to the separate contributions from fluorophores of distinctly different lifetimes which have emission maxima at different wavelengths.
Journal of Fluorescence, 2000
This work presents the development of a method for rapid bacterial identification based on the autofluorescence spectrum. It was demonstrated differences in the autofluorescence spectrum in three bacterial species and the subsequent separation, through the Principal Components Analysis (PCA) technique, in groups with high likeness, that could identify the bacteria in less than 10 min. Fluorescence spectra of 60 samples of 3 different bacterial species (Escherichia coli, EC, Enterococcus faecalis, EF and Staphylococcus aureus, SA), previously identified by automated equipment Mini API, were collected in 10 excitation wavelengths from 330 to 510 nm. The PCA technique applied to the fluorescence spectra showed that bacteria species could be identified with sensitivity and specificity higher than 90% according to differences that occur within the spectra with excitation of 410 nm and 430 nm. This work presented a method of bacterial identification of three more frequent and more clinically significant species based on the autofluorescence spectra in the excitation wavelengths of 410 and 430 nm and the classification of the spectra in three groups using PCA. The results demonstrated that the bacterial identification is very efficient with such methodology. The proposed method is rapid, ease to perform and low cost compared to standard methods.
Journal of Applied Microbiology, 2020
Aims: To provide information on the time dependent behavior of microbe staining by fluorescent dyes in the order of seconds, which is important in terms of the recent rapid and online techniques for microbe measurements and/or environmental microbe analysis. Methods and Results: For combinations of yeast (S. cerevisiae) and typical dyes, including DAPI and Auramine-O, a suspension of yeast cells in ultrapure water was injected into a dye solution in a micro cuvette placed inside a spectrofluorometer and the fluorescence intensity of the resulting solution was measured at one second intervals, starting immediately after the mixing and continued until the time for the maximum intensity using various concentrations of yeast and dyes. The relaxation time , which corresponds to ~63.2% of the maximum fluorescence intensity, was shown to decrease to below 1 second with increasing DAPI concentration while it remained constant for 2-3 seconds with increasing Auramine-O concentration, e.g., at a yeast concentration of 100 µg mL-1. 29 Conclusions: For the conditions of yeast >10 µg mL-1 , DAPI >1 µg mL-1 and Auramine-O >0.1 µg mL-1 , could be adjusted to below 5 seconds to achieve a rapid and stable staining. Impact of the Study: Design and operating conditions for rapid and online measurements of microbes can be optimized.
Analysis of Environmental Samples with Yeast-Based Bioluminescent Bioreporters
Environmental Monitoring, 2011
Environmental Monitoring 4 1.1 Bioreporters Reporter gene fusions have been widely used for the detection and quantification of chemical, biological, and physical agents (Daunert et al., 2000). The principle is to fuse a specific genetic promoter or response element with a reporter gene. Induction by a specific target chemical initiates transcription/translation of the bioreporter molecule, which generates a measurable signal. There are three widely-used classes of bioreporters: colorimetric (e.g. lacZ, cat), fluorescent (e.g. gfp), and bioluminescent (e.g. luc, lux). One example of a colorimetric-based bioreporter is the lacZ gene which encodes thegalactosidase enzyme.-Galactosidase mediates the breakdown of lactose to glucose + galactose. As a bioreporter,-galactosidase is widely used in molecular biology in the bluewhite screening assay. The chromophore X-gal (bromo-chloro-indolyl-galactopyranoside) is cleaved into galactose and an indole moiety that turns the medium blue. For chemical detection, lacZ is fused to a chemical-responsive promoter and when the cells are exposed to chromophores, such as chlorophenol red-D-galactopyranoside (CPRG), the assay medium changes from yellow to red. This type of colorimetric bioreporter is inexpensive and can be used in a qualitative or quantitative type of assay. Color density can be measured on a standard spectrophotometer. Fluorescent assays take advantage of the green fluorescent protein (GFP). GFP was originally isolated from the jellyfish Aequorea victoria (Johnson et al., 1962; Shimomura et al., 1962). GFP is widely used as a bioreporter in eukaryotic systems for its simplicity to clone and no requirement for an organic substrate other than excitation with either UV or blue light. Quantification of the signal is by a fluorescent spectrophotometer or plate reader. There are different versions of gfp including blue-, red-, and yellow-shifted variants each requiring different excitation wavelengths and each of which fluoresce at different wavelengths (Hein & Tsien, 1996; Kendall & Badminton, 1998). In some cases this may be advantageous, especially when multiple bioreporters will be used simultaneously. These genes have been used extensively since they were first employed as gene expression biomarkers (Chalfie et al., 1994). Firefly luciferase is another well-used bioreporter in eukaryotic systems. The luciferase, encoded by the luc gene (lucFF), was originally isolated from Photinus pyralis (firefly) and generates luciferase by a two-step conversion of D-luciferin to oxyluciferin (de Wet et al., 1985). This reaction generates light at 560 nm. However, the gene does not encode for the Dluciferin substrate and therefore substrate addition in any assay is required, which adds processing time and expense to the assay. Luc-based assays may also be constrained by the requirement for a cell lysis step followed by addition of the D-luciferin, adding both time and expense to the assay. Bacterial bioluminescence has been widely used as a bioreporter in prokaryotic systems. The lux operon (luxCDABE) was originally isolated from Vibrio fischeri (Engebrecht et al., 1983), Vibrio harveyi (Cohn et al., 1983), and Photorhabdus luminescens (Szittner & Meighen, 1990). The lux operon encodes for the luciferase enzyme (luxAB) and the long-chain aldehyde substrate (luxCDE) for that reaction. An assay employing bacterial bioluminescence does not require an external organic substrate; the only requirement is for oxygen (O 2). A long chain aldehyde and a reduced flavin mononucleotide (FMNH 2) are converted by luciferase (LuxAB) to a long chain carboxylic acid and FMN, producing light at 490 nm wavelength (Meighen & Dunlap, 1993). The luxAB (without luxCDE) can also be used as a bioreporter and while these strains also produce light at 490 nm, they are less suited for high www.intechopen.com
Bioluminescent System of Luminous Bacteria for Detection of Microbial Contamination
Journal of Siberian Federal University. Biology, 2018
Microbial contamination is usually analyzed using luciferin-luciferase system of fireflies by the detection of adenosine-5'-triphosphate (ATP). There is an opportunity to assess the bacterial contamination of various objects based on a quantitative analysis of other nucleotides. In the present study, a bioluminescent enzyme system of luminous bacteria NADH:FMN-oxidoreductase (Red) and luciferase (BLuc) was investigated to understand if it can be used for quantitative measurements of bacterial cells by nicotinamide adenine dinucleotide (NADH) and flavin mononucleotide (FMN) detection. To increase the sensitivity of bioluminescent system to FMN and NADH, optimization of assay conditions was performed by varying enzymes and substrates concentrations. The lowest limits of detection were 1.2 nM FMN and 0.1 pM NADH. Escherichia coli cells were used as a model bacterial sample. FMN and NADH extraction was made by destructing cell membrane by ultrasonication. Cell suspension was added into the reaction mixture instead of FMN and NADH, and light intensity depended on number of bacterial cells in the reaction mixture. Centrifugation of sonicated sample as an additional step of sample preparation did not improve the sensitivity of method. The experimental results showed that Red and BLuc system could detect at least 800 thousand bacterial cells mL-1 by determining concentration of NADH extracted from lysed cells, while 3.9 million cells mL-1 can be detected by determining concentration of FMN.
Applications of Biochemiluminescence in Quality Assurance of Food Products
To attend the food industries demands, rapid and sensitive methods have been developed to evaluate microbiological quality of several foods and water. The cleanliness of the environment in which foods are processed and stored must be monitored. Newly refined testing methods allow rapid monitoring and detection of bacteria, pinpointing any sources of contamination in food handling, storing and processing. Bioluminescence is light produced by a chemical reaction within an organism. The ATP bioluminescence assay (test) is based on the fact that all living cells contain adenosine triphosphate (ATP). ATP bioluminescence is not a microbial count method but it is sensitive enough to detect the ATP content of individual cells in small numbers. ATP is determined by using its reaction with luciferin, a light-emitting molecule found in fireflies.