Simple quantification of bacterial envelope-associated extracellular materials

We have developed a simple method for distinguishing between bacterial cultures that produce different amount of exopolysaccharide. It is based upon small differences in pellet volume formed by those cultures upon centrifugation. For that we have constructed a special centrifugation tube consisting of two connected chambers: an upper 12 ml chamber connected to a lower capillary chamber. Cells are applied to the upper chamber and following centrifugation, sink to its bottom and are forced into the capillary so that the height they fill can be measured. This procedure has been developed in order to demonstrate differences in volume of centrifugation pellet formed by similar number of Escherichia coli K12 wild type, rpoS mutant and yjbG rpoS double mutant cells. These differences are further shown to be a result of overproduction of colanic acid exopolysaccharide in the mutant strains. We suggest that this simple method can be employed to detect differences in other cell surface structures and to estimate biomass when optical density measurement or microscopic count is not applicable.     

Simple and sensitive determination of five quinolones in food by liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of five different quinolones: enrofloxacin, ciprofloxacin, sarafloxacin, oxolinic acid and flumequine in pork and salmon muscle. The method includes one extraction and clean-up step for the five quinolones together which are detected in two separated HPLC runs by means of their fluorescence. The proposed analytical method involves homogenizing of the tissue sample with 0.05 M phosphate buffer, pH 7.4 and clean-up by Discovery DS-18 cartridges. For chromatographic separation a Symmetry C(18) column is used in two different runs: (1) ciprofloxacin, enrofloxacin and sarafloxacin with acetonitrile-0.02 M phosphate buffer pH 3.0 (18:82) as mobile phase and the detector at excitation wavelength: 280 nm and emission wavelength 450 nm; and (2) oxolinic acid and flumequine with acetonitrile-0.02 M phosphate buffer pH 3.0 (34:66) as mobile phase and excitation wavelength: 312 nm and emission wavelength: 366 nm. Detection limit was as low as 5 ng g(-1), except for sarafloxacin which had a limit of 10 ng g(-1). Standard curves using blank muscle tissues spiked at different levels showed a good linear correlation coefficient, r(2) higher than 0.999 for all quinolones.     

Dissecting DNA-protein and protein-protein interactions involved in bacterial transcriptional regulation by a sensitive protein array method combining a near-infrared fluorescence detection

The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region. Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions). Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach. We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E. coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP. We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E. coli RNA polymerase alpha subunit.     

Development and validation of a simple, sensitive enzyme immunoassay (EIA) for quantification of prolactin in buffalo plasma

A simple and highly sensitive enzyme immunoassay (EIA) was developed and validated for prolactin quantification in buffalo plasma (on a microtitreplate) using the biotin-streptavidin-peroxidase amplification and immobilized antiserum in a competitive assay. Prolactin standards (range: 5-5000 pg/(well 50 microL)) were prepared in hormone-free plasma collected from minimal stress non-lactating buffalo heifers in temperate weather. The sensitivity of the EIA procedure was 5 pg/(well 50 microL) (corresponds to 0.1 ng/mL plasma); the 50% relative binding sensitivity occurred at 160 ng/(well 50 microL). Plasma volumes for the EIA, viz. 12.5, 25, and 50 microL, did not influence the shape of standard curve. A parallelism test was carried out to compare the endogenous buffalo plasma prolactin with bovine prolactin standard. To validate the assay biologically, 11 Murrah buffaloes were given a third-generation antiprolactin (Norprolac; 10 mg/animal, i.m.). Blood samples were collected 1 d prior to the start of Norprolac administration and continued up to seventh day in an Ovsynch treatment program. In all animals, there were abrupt declines in prolactin concentrations following Norprolac treatments, which confirmed the biological validation of the EIA. After development and validation of EIA procedure, the concentration of plasma prolactin was determined efficiently in samples collected during both summer and winter samples.     

Simple, sensitive and on-line fluorescence monitoring of photodegradation of phenol and 2-naphthol.

A simple molecular fluorescence spectrometer based on a hand-held CCD spectrometer was constructed for on-line monitoring of the photodegradation of pollutants. A high-pressure Hg vapour lamp was used for the UV photodegradation and simultaneously for the fluorescence excitation. Phenol and 2-naphthol were selected as the targets for this preliminary study. Using peak fluorescence, figures of merit for monitoring these two hydroxybenzene were obtained. Degradation efficiencies with different homogeneous photocatalyst systems were investigated, including UV only, UV/H(2)O(2) and UV/Fe(3+) degradation systems. The kinetics modelling showed that their photodegradation fitted the Langmuir-Hinshelwood model. Results showed that the proposed method is potentially applicable to both on-line real-time monitoring and field analysis.     

Rapid and simple quantification of bacterial cells by using a microfluidic device

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.     

Simple and sensitive method for the determination of celecoxib in human serum by high-performance liquid chromatography with fluorescence detection

A simple method is described for the determination of the cyclooxygenase-2 specific inhibitor celecoxib in human serum by HPLC using the demethylated analogue as internal standard. After protein precipitation with acetonitrile, samples were extracted with chloroform. Separation was achieved on a Prontosil C18 AQ column (150x3 mm I.D., 3-microm particle size) at a flow-rate of 0.35 ml/min using water-acetonitrile (40:60, v/v) as the mobile phase. Using fluorescence detection with excitation at 240 nm and emission at 380 nm, the limit of quantification was 12.5 ng/ml for a sample size of 0.5 ml of serum. The assay was linear in the concentration range of 12.5-1500 ng/ml and showed good accuracy and reproducibility. At all concentrations intra- and inter-assay variabilities were below 11% with less than 9% error. The method was applied to the determination of celecoxib for pharmacokinetic studies in man.     

Simple and sensitive procedure for the assay of serotonin and catecholamines in brain by high-performance liquid chromatography using fluorescence detection

A simple, rapid and specific method for the determination of serotonin and catecholamines in brain is described. After tissue homogenisation, catecholamines are isolated by adsorption onto alumina and elution with perchloric acid. Serotonin is isolated by extraction into n-heptanol and back-extraction into acid. High-performance liquid chromatography of the acid extracts is performed with a C₁₈ reversed-phase column and simple mobile phases. Detection is by the intrinsic fluorescence of the amines on excitation at 200 nm. Detection limits are 100 pg for norepinephrine, 300 pg for dopamine and 20 pg for serotonin. The results are found to correlate well with a catechol O-methyl transferase radioenzymatic assay for catecholamines and a ninhydrin derivatisation procedure for serotonin.     

Microscopic quantification of bacterial invasion by a novel antibody-independent staining method

Microscopic discrimination between extracellular and invasive, intracellular bacteria is a valuable technique in microbiology and immunology. We describe a novel fluorescence staining protocol, called FITC-biotin-avidin (FBA) staining, which allows the differentiation between extracellular and intracellular bacteria and is independent of specific antibodies directed against the microorganisms. FBA staining of eukaryotic cells infected with Gram-negative bacteria of the genus Neisseria or the Gram-positive pathogen Staphylococcus aureus are employed to validate the novel technique. The quantitative evaluation of intracellular pathogens by the FBA staining protocol yields identical results compared to parallel samples stained with conventional, antibody-dependent methods. FBA staining eliminates the need for cell permeabilization resulting in robust and rapid detection of invasive microbes. Taken together, FBA staining provides a reliable and convenient alternative for the differential detection of intracellular and extracellular bacteria and should be a valuable technical tool for the quantitative analysis of the invasive properties of pathogenic bacteria and other microorganisms.     

Electrochemiluminescence of peroxydisulfate enhanced by L-cysteine film for sensitive immunoassay

A novel label free electrochemiluminescence (ECL) immunosensor based on the ECL of peroxydisulfate solution for detection of α-1-fetoprotein (AFP) has been developed. For this proposed immunosensor, L-cysteine was firstly electrodeposited on the gold electrode surface, which promoted the electron transfer and largely enhanced the ECL of peroxydisulfate solution. Subsequently, gold nanoparticles (nano-Au) were assembled onto the L-cysteine film modified electrode to improve the absorption capacity of antibody and further amplify the ECL signal. Then, antibody was immobilized onto the electrode through nano-Au. At last bovine serum albumin (BSA) was employed to block the nonspecific binding sites. As a result, a novel ECL immunosensor was firstly obtained by applying the ECL of peroxydisulfate solution without conventional luminescent reagents. The AFP was determined in the range of 0.01-100 ng mL(-1), with a low detection limit of 3.3 pg mL(-1) (S/N=3). The proposed ECL immunosensor provides a rapid, simple, and sensitive immunoassay protocol for protein detection, which might hold a promise for clinical application. Moreover, this work would open up a new field in the application of peroxydisulfate solution ECL for highly sensitive bioassays.     

A high throughput Nile red fluorescence method for rapid quantification of intracellular bacterial polyhydroxyalkanoates

A rapid quantitative measurement of accumulated polyhydroxyalkanoate (PHA) is essential for rapid monitoring of PHA production by microorganisms. In the present study, a 96-well microplate was used as a high throughput means to measure the fluorescence intensity of the Nile red stained cells containing PHA. The linear correlation obtained between intracellular PHA concentration and the fluorescence intensity represents the potential of the Nile red method employment to determine PHA concentration. The optimal ranges of excitation and emission wavelengths were determined using bacterial cells containing different types of PHAs, of different co-monomers and compositions. Interestingly, in spite of different co-monomers compositions in each PHA, all tested PHAs fluoresced maximally at excitation wavelength between 520 and 550 nm, and emission wavelength between 590 and 630 nm. The developed staining method also had successfully demonstrated a good correlation between the amount of accumulated PHA based on the fluorescence intensity measurements and that from chromatographic analysis to evaluate poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)], using the same calibration curve, despite of different co-monomers that the PHA consist. Strongly supported by these experimental results, it can therefore be concluded that the developed staining method can be efficiently applied for rapid monitoring of PHA production.     

Simple and sensitive method for quantification of low tobramycin concentrations in human plasma using HPLC-MS/MS

After oral administration of tobramycin, as part of selective decontamination of the digestive tract (SDD) in critically ill patients, absorption of tobramycin from the gut into the blood may take place. To quantify low concentrations of tobramycin in human plasma, we developed and validated a simple (sample pre-treatment consisting of protein precipitation with acetonitrile using 200microl plasma), rapid (runtime 3min using a Pathfinder MR reversed-phase column) and sensitive (concentration range of 0.05-1.0mg/l using MS/MS detection) method.     

Comparison of fluorescence and resonance light scattering for highly sensitive microarray detection of bacterial pathogens

Microarrays have emerged as potential tools for bacterial detection and identification. Given their high parallelism, they might represent a breakthrough in current diagnostic methods, provided they can be coupled to simplified labeling protocols and detected with adequate sensitivities. We describe here a technique to directly label total bacterial RNA, thus avoiding the multiple steps and possible biases associated with enzymatic amplification (e.g. PCR). We have then compared the performances of one white-light source and two laser-based fluorescence scanners for detection reliability and sensitivity. Our study reveals that nanoparticle-labeled bacterial RNA generates reproducible resonance light scattering signals that are at least 50 times more intense than state-of-the-art confocal-based fluorescence signals.     

Determination of human tumor necrosis factor alpha by a highly sensitive enzyme immunoassay.

Tumor necrosis factor alpha (TNF-alpha) is a polypeptide produced primarily by monocytes and macrophages. It is involved in a wide variety of immune reactions. A simple and sensitive microplate enzyme-linked immunosorbent assay for the detection of hTNF-alpha in serum, plasma, and cell culture supernatants is described. The method is based on the use of horseradish peroxidase in biotin-streptavidin amplification system which is performed in Nunc StarWell. This system has enabled us to achieve a sensitivity of 0.1 pg hTNF-alpha/ml of the sample. The assay is calibrated to the World Health Organization (WHO) standard for hTNF-alpha (87/650). The within-run coefficient of variation ranged from 3.7 to 5.9 and the between-run coefficient of variation ranged from 8.0 to 9.9. The results obtained by the proposed method and by a commercially available kit (DRG hTNF-alpha ELISA) correlated well (n = 20, r = 0.956).     

Practical applications in molecular biology of sensitive fluorescence detection by a laser-excited fluorescence image analyzer.

A new kind of fluorescence image analyzer was developed for a variety of uses, especially in molecular biology. Compounds labeled with fluorescent groups on a gel or nitrocellulose membrane are excited with 532 nm of light from a green laser. The fluorescence emitted passes through light-collecting fibers to a photomultiplier. Imaging data converted from the emitted light are analyzed by a microcomputer and stored on a magnetic optical disk. Dideoxy DNA sequencing was done with the same amount of DNA used for autoradiography, and the sequencing ladders obtained from gel scanning were automatically converted to sequence data by the analyzer. When an agarose gel was analyzed after electrophoresis, DNA stained with ethidium bromide was detected by the analyzer with higher sensitivity rather than by the conventional photographic method. Nylon and nitrocellulose membranes could be read by the analyzer, so blot hybridization experiments can be done without radioisotopes. High-quality computer storage of the imaging data from gel electrophoresis and hybridized membranes, including pulsed-field gels, make it possible to quantify image intensity and to construct many kinds of databases.     

Variations in the fluorescence intensity of intact DAPI-stained bacteria and their implications for rapid bacterial quantification

J. ROSS, P.I. BOON, R. SHARMA AND R. BECKETT. 1996. As current techniques for the quantification of bacteria are laborious and often imprecise, instrumental approaches such as sedimentation field-flow fractionation (SdFFF) are attractive. In this technique, fluorogenic dyes specific for nucleic acids are used to identify bacterial cells. Bacterial biomass can be quantified directly with SdFFF if the specific fluorescence of bacterial cells is constant. The effect of different growth conditions on the specific fluorescence of one strain each of Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Staphylococcus epidermidis stained with 4',6-diamidino-2-phenylindole was examined. Specific fluorescence varied over a 500-fold range, from 0.22 to 103 arbitrary fluorescence units per cell. Specific fluorescence was highest when cells were in log phase, and lowest when cells were in stationary phase. Specific fluorescence decreased when cells harvested in log phase were starved for 7 d in a carbon-free minimal medium, and increased rapidly (within 2 h) after cells were relieved from carbon limitation. Such variations in specific fluorescence must be considered when using gross fluorescence as a direct indicator of bacterial numbers in the SdFFF technique for quantifying bacterial biomass. Moreover, they have serious implications for the application of fluorescence techniques in other instrumental approaches for bacterial enumeration in environmental samples.     

Rapid and sensitive detection of 17beta-estradiol in environmental water using automated immunoassay system with bacterial magnetic particles

A fully automated immunoassay of 17beta-estradiol (E2) was performed using anti-E2 monoclonal antibody immobilized on bacterial magnetic particles (AntiE2-BMPs) and alkaline phosphatase-conjugated E2 (ALP-E2). E2 concentration in environmental water samples was evaluated by decrease in luminescence based on competitive reaction. A linear correlation between the luminescence intensity and E2 concentration was obtained between 0.5 and 5 ppb. The minimum detectable concentration of E2 was 20 ppt. All measurement steps were done within 0.5 h. The analysis of environmental water samples by a commercially available ELISA kit and the BMP-based immunoassay gave good correlation plots with a correlation efficient of 0.992. These results suggest that the fully automated system using the BMP-based immunoassay has some advantages in the high rapidity and sensitivity of the measurement. This system will enable us to determine low E2 concentrations without sample condensation.