Raman spectroscopic identification of single bacterial cells at different stages of their lifecycle

Early, rapid, and reliable bacterial identification is of great importance in natural environments and in medical situations. Numerous studies have shown that Raman spectroscopy can be used to differentiate between different bacteria under controlled laboratory conditions. However, individual bacteria within a population exhibit macromolecular and metabolic heterogeneity over their lifetime. Therefore it is important to be able to identify and classify specific bacteria at different time points of the growth cycle. In this study, four species of bacteria were used to explore the capability of confocal Raman spectroscopy as a tool for the identification of (and discrimination between) diverse bacterial species at various growth time points. The results show that bacterial cells from different growth time points (as well as from a random growth phase) can be discriminated among the four species using principal component analysis (PCA). The results also show that bacteria selected from different growth phases can be classified with the help of a prediction model based on principal component and linear discriminant analysis (PC-LDA). These findings demonstrate that Raman spectroscopy with the application of a PC-LDA model rooted in chemotaxonomic analysis has potential for rapid sensing of microbial cells in environmental and clinical studies.     

Raman spectroscopic identification of single bacterial cells under antibiotic influence

The identification of pathogenic bacteria is a frequently required task. Current identification procedures are usually either time-consuming due to necessary cultivation steps or expensive and demanding in their application. Furthermore, previous treatment of a patient with antibiotics often renders routine analysis by culturing difficult. Since Raman microspectroscopy allows for the identification of single bacterial cells, it can be used to identify such difficult to culture bacteria. Yet until now, there have been no investigations whether antibiotic treatment of the bacteria influences the Raman spectroscopic identification. This study aims to rapidly identify bacteria that have been subjected to antibiotic treatment on single cell level with Raman microspectroscopy. Two strains of Escherichia coli and two species of Pseudomonas have been treated with four antibiotics, all targeting different sites of the bacteria. With Raman spectra from untreated bacteria, a linear discriminant analysis (LDA) model is built, which successfully identifies the species of independent untreated bacteria. Upon treatment of the bacteria with subinhibitory concentrations of ampicillin, ciprofloxacin, gentamicin, and sulfamethoxazole, the LDA model achieves species identification accuracies of 85.4, 95.3, 89.9, and 97.3 %, respectively. Increasing the antibiotic concentrations has no effect on the identification performance. An ampicillin-resistant strain of E. coli and a sample of P. aeruginosa are successfully identified as well. General representation of antibiotic stress in the training data improves species identification performance, while representation of a specific antibiotic improves strain distinction capability. In conclusion, the identification of antibiotically treated bacteria is possible with Raman microspectroscopy for diverse antibiotics on single cell level.

Micro-Raman spectroscopic identification of natural mineral phases and their weathering products inside an abandoned zinc/lead mine

Mining activities provide a good source of minerals of different nature. On the one hand, the primary minerals for whose formation a geological time-scale is required. On the other hand, secondary minerals, formed from removed products after the earlier weathering and alteration states. These are characteristic of the local geology and the environment context that commonly appears due to the low chemical stability of their original primary minerals. This work shows how quickly the reactions promoting secondary minerals may have taken place, due to the fact that these were found in newly formed solid materials called efflorescences. To achieve this purpose, the sampling is crucial. It was carried out in such a way that tried to guarantee that the samples collected consisted in the very top soil matter (first 2 cm depth). Thus, unlike the deeper soil, the material analysed may have been newly formed due to the interactions that they had with the place weathering agents (i.e. air oxygen, humidity, and microbial activities). Raman spectroscopy has emerged as a good and fast non-destructive technique that provides molecular information of the local mineralogy without the need of any pre-treatment of the samples. At the same time, the work looked for information on the variety of non-stable lead and-or zinc containing minerals due to the possible health and environmental risks they convey. Among the different minerals identified, 16 were of primary nature while 23 may be classified as secondary minerals, probably formed in the last decades as the result of the extractive activities.     

Vibrational spectroscopic identification of isoprene,pinenes and their mixture

Here we show a study of vibrational spectroscopic identification of a few typical organic compounds which are known as the main sources of organic aerosols(OAs) particle matter in air pollution. Raman and IR spectra of isoprene, terpenoids, pinenes and their mixture are meticulously examined, showing distinguishable intrinsic vibrational spectroscopic fingerprints for these chemicals, respectively. As a reference, first-principles calculations of Raman and infrared activities are also conducted. It is interestingly found that, the experimental spectra are peak-to-peak consistent with the DFT(Density Functional Theory)-calculated vibrational activities. Also found is that, in a certain case such as for bpinene, a dimer model, rather than an isolated single molecular model, reproduces the experimental results, indicating unneglected intermolecular interactions. Starting with this study, we are endeavoring to advocate a database of Raman/IR fingerprint spectra for OA haze identification.     

Chromatographic and spectroscopic methods for the identification of alkaloids from herbarium samples of the genus Uncaria.

A combination of thin-layer chromatography, gas-liquid chromatography, ultraviolet spectroscopy and mass spectrometry techniques for the alkaloid screening of herbarium samples of the genus Uncaria (Rubiaceae) is described. Some sixty alkaloids are distinguished by the screening procedure, and they represent heteroyohimbine, oxindole, roxburghine, simple beta-carboline, pyridino-indolo-quinolizidinone and gambirtannine types.     

Fourier-transform infrared spectroscopic study of the interactions of selenium species with living bacterial cells

A study of the interactions of several selenium species with living bacterial cells was carried out by Fourier-transform infrared (FT-IR) spectroscopy. Bacterial cells consisted of an Escherichia coli strain (K-12) cultivated in a growth medium based on glucose contaminated with selenium species. Equilibrium between the analyte in the solution and the extraction medium was established, and then the effects of selenium species upon the external membrane of the living bacterial cells were characterized by performing FT-IR spectroscopy of whole cells. The presence of the toxicants at various concentrations in the culture medium had an effect on the FT-IR spectra, and the concentration of the selenium species was determined directly in the biomass by FT-IR spectroscopy. The intensity ratios between several absorption lines, which varied as a function of the concentration of the selenium species, were used as the analytical signal.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Combined spectroscopic and topographic characterization of nanoscale domains and their distributions of a redox protein on bacterial cell surfaces

Redox protein nanoscale domains on the cell surface of a bacterium, Shewanella oneidensis MR1, grown in the absence and presence of electron acceptors, is topographically characterized using combined atomic force microscopy (AFM) and confocal surface enhanced Raman scattering (SERS) spectroscopy. The protruding nanoscale domains on the outer membrane of S. oneidensis were observed, as was their disappearance upon exposure to electron acceptors such as oxygen, nitrate, fumarate, and iron nitrilotriacetate (FeNTA). Using SERS spectroscopy, a redox heme protein was identified as a major component of the cell surface domains. This conclusion was further confirmed by the disappearance of Raman vibrational frequencies, characteristic of heme proteins, upon exposure of the cells to electron acceptors. Our experimental results from our AFM imaging and SERS spectroscopy, consistent with the literature, suggest the protruding nanoscale surface domains as heme-containing secretions. Our results on the distributions of redox proteins on microbial cell surfaces will be helpful for a mechanistic understanding of the behaviors of surface proteins and their interactions with redox environments.     

Vibrational spectroscopic study on pH dependence of some diazines

The infrared and Raman spectra of several pyrazines and pyridazines were measured in a wide acidity range. Methods of measurements were elaborated for extreme acidity conditions. The approximate assignments of some spectra were determined. The spectra show bands with pH depending frequencies and intensities and give information on the solute—solvent interaction.     

Fluorescent DNA chemosensors: identification of bacterial species by their volatile metabolites

Polyfluorophores built on a DNA scaffold (ODFs) were synthesized and tested for fluorescence responses to the volatiles from M. tuberculosis, E. coli and P. putida in closed Petri dishes. Two sensors in a pattern-based response could distinguish the bacterial strains accurately, suggesting the use of ODFs in rapid identification of infectious agents.     

Modeling dependence of the strength of agglomerates on their basicity

An attempt was performed to clarify and summarize mathematically an extreme dependence strength-basicity of agglomerate based on polymorphism of Ca₂SiO₄. Two main indicators underlaid the sinter production: a mechanical strength and specific performance of a sinter plant, which can be expressed by yield of a certain size fractions after physical stress and by linear sintering rate, respectively.     

Trapping and spectroscopic identification of the photointermediates of bacteriorhodopsin at low temperatures

Light-driven transmembrane proton pumping by bacteriorhodopsin occurs in the photochemical cycle, which includes a number of spectroscopically identifiable intermediates. The development of methods to crystallize bacteriorhodopsin have allowed it to be studied with high-resolution X-ray diffraction, opening the possibility to advance substantially our knowledge of the structure and mechanism of this light-driven proton pump. A key step is to obtain the structures of the intermediate states formed during the photocycle of bacteriorhodopsin. One difficulty in these studies is how to trap selectively the intermediates at low temperatures and determine quantitatively their amounts in a photosteady state. In this paper we review the procedures for trapping the K, L, M and N intermediates of the bacteriorhodopsin photocycle and describe the difference absorption spectra accompanying the transformation of the all-trans-bacteriorhodopsin into each intermediate. This provides the means for quantitative analysis of the light-induced mixtures of different intermediates produced by illumination of the pigment at low temperatures.     

Influence of measuring conditions on the quantification of spectroscopic signals in TA-FTIR-MS systems

Simultaneous thermal analysis (TA) and evolved gas analysis by mass spectrometry (MS) and/or Fourier transform infrared spectroscopy (FTIR) is a powerful hyphenated technique combining direct measurement of mass loss and sensitive spectroscopic analysis. In the present study the influence of several experimental parameters which may affect the quantification of FTIR signals have been studied using a combined TA-FTIR-MS system. Parameters studied include: sample mass (1-400 mg), carrier gas flow rate (25-200 mL min^-1), resolution of the FTIR spectrometer (1-32 cm^-1), and location of injection of the calibrating gas. MS analysis, which was not significantly affected by the experimental conditions, was used as a reference for assessing the accuracy of quantification by FTIR. The quantification of the spectroscopic signals was verified by the decomposition (NaHCO₃) or dehydration (CuSO₄·5H₂O) of compounds with well-known stoichiometry. The systematic study of the parametric sensitivity revealed that spectral resolution and carrier gas flow rate, which affect the acquisition time in the IR-cell, are key parameters that must be adjusted carefully for reliable quantification. The dependence of the reliability of quantification on these parameters is illustrated and conditions leading to proper quantification are discussed. As an example, for a standard spectral resolution of 4 cm^-1 and a FTIR gas cell volume of 8.7 mL, the carrier gas flow must be lower than 100 mL min^-1 for warranting accurate results (relative deviation <2%). The concentration range of analyzed species is limited but can be extended by proper selection of the wavenumber regions for molecules giving strong IR signals.     

Concentration dependence of the spectroscopic and photochemical properties of atomic and molecular oxygen in argon matrices

Vacuum ultraviolet (VUV) excitation (200-100 nm) and visible emission (300-650 nm) spectra of O2 imbedded in Ar matrices at different concentrations are presented. At 0.1 and 0.2% concentrations a linear increase in the intensities of the excitation and emission spectra is observed. At these concentrations, photolysis of O2 is found to be negligible. At higher concentrations (0.5, 1 and 2%) the normalized intensities of the excitation and emission spectra of O2 decrease. With increasing concentration of O2 the permanent photolysis of O2 increases, which does not correlate, based on the excitation spectra of O in Ar, to the production of isolated O atoms. It has been shown that this anomalous behavior should be due to the formation of van der Waals dimers and oligomers at higher concentrations of O2 that upon photolysis produce ozone.     

Recent studies on advance spectroscopic techniques for the identification of microorganisms: A review

The continuous development of resistance to antibiotic drugs by microorganisms causes high mortality and morbidity. Pathogens with distinct features and biochemical abilities make them destructive to human health. Therefore, early identification of the pathogen is of substantial importance for quick ailments and healthcare outcomes. Several phenotype methods are used for the identification and resistance determination but most of the conventional procedures are time-consuming, costly, and give qualitative results. Recently, great focus has been made on the utilization of advanced techniques for microbial identification. This review is focused on the research studies performed in the last five years for the identification of microorganisms particularly, bacteria using advanced spectroscopic techniques including mass spectrometry (MS), infrared (IR) spectroscopy, Raman spectroscopy (RS), and nuclear magnetic resonance (NMR) spectroscopy. Among all the techniques, MS techniques, particularly MALDI-TOF/MS have been widely utilized for microbial identification. A total of 44 bacteria i.e., 6 Staphylococcus spp., 3 Enterococcus spp., 6 Bacillus spp., 4 Streptococcus spp., 6 Salmonella spp., and one from each genus including Escherichia, Acinetobacter, Pseudomonas, Proteus, Clostridioides, Candida, Brucella, Burkholderia, Francisella, Yersinia, Moraxella, Vibrio, Shigella, Serratia, Citrobacter, and Haemophilus (spp.) were discussed in the review for their identification using the above-mentioned techniques. Among all the identified microorganisms, 21% of studies have been conducted for the identification of E. coli, 14% for S. aureus followed by 37% for other microorganisms.     

FT-Raman spectroscopic studies of metal oxalates and their mixtures

The Raman spectra of a number of Group I and II metal oxalates are presented. From quantitative spectroscopic studies of the pure compounds and of mixtures of calcium, potassium and magnesium oxalates, it has been demonstrated that each component can be determined in the prepared mixtures to better than 2%. Comparisons made with the Raman spectra of lithium and sodium oxalates indicate that there are characteristic differences in the observed spectra of the Group I metal oxalates and that several vibrational bands of C₂O²⁻₄ are sensitive to the cation, being shifted to lower wavenumbers as the cation mass increases from Li to K.     

Enthalpies of formation of methoxybenzoic acids and their dependence on structure

The energy of combustion of 2,5-dimethoxybenzoic acid has been determined using a static bomb calorimeter. The vapor pressures of the compound have been measured over a 18 K temperature interval by the Knudsen effusion technique. Heat capacity measurements betweenT=270 K andT=338 K were carried out by DSC. From these experimental results the standard molar enthalpies of combustion, sublimation, and formation in the crystalline and gaseous state at the temperature 298.15 K have been derived. With this compound, the series of mono- and dimethoxy-benzoic acids have been completed. Their_f H _m ^o values were expressed by an additive relationship, taking into account the number of methoxy groups and the number of all 1,2 interactions: an accuracy of 3.3 kJ·mol^–1 was achieved. In an alternative approach the substituent effect of the methoxy groups was evaluated within the framework of isodesmic reactions. The effect of disubstitution was referred to mono derivatives and the excess energy—the so-called buttressing effect—was evaluated (2–24 kJ· mol^–1 for individual bis derivatives). These values were explained in terms of the conformation of the methoxy group around the C_ar-O bond.     

Systematic study of the photoluminescence dependence of thiol-capped CdTe nanocrystals on the reaction conditions

A modified method to prepare high-quality thiol-capped CdTe nanocrystals (NCs) was reported in this paper. The experimental results showed that the different molar ratios of the ligands (thioglycolic acid) to monomers (Cd2+ ions) in the precursor solution played an important role in the photoluminescence (PL) quantum yield (QY) of the as-prepared CdTe NCs. When [ligand]/[monomer] = 1.2, the maximum fluorescent emission peak appeared in the orange-red window, and the PL QY increased up to 50% at room temperature without any postpreparative treatment. In the meantime, suitable reaction conditions were in favor of the optimization of the surface structure of NCs, resulting in the relatively high PL QY from green to red. In addition, some differences between hydrothermal synthesis and traditional aqueous synthesis of CdTe NCs were discussed.     

H-C-SiH3: direct generation and spectroscopic identification of ethylidene's cousin

Ground-state triplet silaethylidene, generated directly by the reaction of 3P carbon atoms with silane under matrix isolation conditions in solid Ar (10-12 K), has been thoroughly characterized by the EPR and IR spectra of both the parent and perdeuterated isotopologs. A theoretical anharmonic vibrational analysis based on a CCSD(T)/cc-pVTZ complete quartic force field gave remarkable agreement with the experimental IR fundamentals, generally within 10 cm-1 and without any empirical scaling of the ab initio frequencies. Silaethylidene exhibits a Cs minimum with a H-C-Si angle near 153 degrees , but the barrier to H-C-Si linearity (C3v symmetry) is only 0.24 kcal mol-1. This minuscule barrier can be surmounted by zero-point vibrations, as evident from the EPR data. The triplet stabilizing effect of the electropositive SiH3 group amounts to about 15 kcal mol-1.