A rapid method for the enumeration of coliforms in processed foods by ion mobility spectrometry

The number of coliforms in processed food was enumerated rapidly by the use of ion mobility spectrometry. This method measured the time to observe o -nitrophenol liberated from the substrate o -nitrophenylgalactoside and was inversely proportional to the logarithm of the initial coliform count. The presence or absence of coliforms was determined within 12 h. This assay was unsuitable for non-processed foods due to the presence of an endogenous β-galactosidase enzyme.     

A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite

The measurement of elemental selenium (Se⁰) is needed to assess the rate and magnitude of bacteria reduction of selenite or selenate. We have developed a spectrophotometric method for the measurement Se⁰ that is rapid and can be employed to measure the quantity of Se⁰ produced by bacterial cultures. This method employs the use of 1 M Na₂S to convert the insoluble elemental selenium to a red-brown solution and with this method there is a direct correlation between concentration of elemental selenium and the absorption at 500 nm. To demonstrate the utility of this assay, we have followed the reduction of selenite to Se⁰ by Moraxella bovis, and by bacterial consortia in soil and water samples.     

A novel method for the measurement of in vitro fatty acid 2-hydroxylase activity by gas chromatography-mass spectrometry

Fatty acid 2-hydroxylase (FA2H), encoded by the FA2H gene, is an enzyme responsible for the de novo synthesis of sphingolipids containing 2-hydroxy fatty acids. 2-Hydroxy sphingolipids are highly abundant in the brain, as major myelin galactolipids (galactosylceramide and sulfatide) contain a uniquely high proportion ( approximately 50%) of 2-hydroxy fatty acids. Other tissues, such as epidermis, epithelia of the digestive tract, and certain cancers, also contain 2-hydroxy sphingolipids. The physiological significance of the 2-hydroxylation on N-acyl chains of subsets of sphingolipids is poorly understood. To study the roles of FA2H and 2-hydroxy sphingolipids in various tissues, we developed a highly sensitive in vitro FA2H assay. FA2H-dependent fatty acid 2-hydroxylation requires an electron transfer system, which was reconstituted in vitro with an NADPH regeneration system and purified NADPH:cytochrome P-450 reductase. A substrate [3,3,5,5-D(4)]tetracosanoic acid was solubilized in alpha-cyclodextrin solution, and the 2-hydroxylated product was quantified by gas chromatography-mass spectrometry after conversion to a trimethylsilyl ether derivative. When the microsomes of FA2H-transfected COS7 cells were incubated with the electron transfer system and deuterated tetracosanoic acid, deuterated 2-hydroxy tetracosanoic acid was formed in a time- and protein-dependent manner. With this method, FA2H activities were reproducibly measured in murine brains and tissue culture cell lines.     

BAC system: A novel method for manipulation of herpesvirus genomes based on bacterial genetics

Although methods for reverse genetics of herpesviruses have been established in early 1980s, the steps are laborious and time-consuming. In 1997, Dr. Koszinwski's group reported a novel approach for the construction of herpesvirus mutants, based on cloning the viral genome as a bacterial artificial chromosome (BAC) in E. coli. This technique allows the maintenance of viral genomes as plasmid in E. coli and the reconstitution of viral progeny by transfection of the BAC plasmid into eukaryotic cells. Any genetics modification of the viral genome in E. coli using bacterial genetics is possible, thereby facilitating the introduction of mutagenesis into herpesvirus genome. This 'BAC system' has opened new avenues for reverse and forward genetics of herpesviruses in basic research and in vector development for human therapy. Here we describe the principle of the 'BAC system' in herpesvirus researches.     

A novel force field parameter optimization method based on LSSVR for ECEPP

In this paper, we propose a novel force field parameter optimization method based on LSSVR and optimize the torsion energy parameters of ECEPP force field. In this method force field parameter optimization problem is turned into a support vector regression problem. Protein samples for regression model training are chosen from Protein Data Bank. The experiments show that the optimized force-field parameters make both α-helix and β-hairpin structures more consistent with the experimental implications than the original parameters.     

A rapid method for the measurement of bacterial chemotaxis

A rapid method for bacterial chemotaxis in a spatial gradient of chemoeffectors has been developed. The method is based on turbidimetric measurement of bacteria movement in a chemoeffector gradient created in a spectrophotometer cuvette. The method was verified onEscherichia coli mutants with known chemotactic properties.     

A simple method for concentration of biogenic amines and their metabolites from biological samples for analysis by HPLC-EC

This report describes a new rapid, convenient and inexpensive method of concentrating biogenic amines and their metabolites from biological samples for analysis by HPLC-EC. Recovery of standard monoamines and metabolites from artificial cerebrospinal fluid (CSF) solution following lyophilization in the presence of glutathione (1.2 mg/ml, final concentration) and EGTA (1.8 mg/ml, final concentration) was greater than 89%; the coefficient of variation was 0.6-3.7%, depending on the specific amine or metabolite concentrated. Lyophilization as a one step procedure is suitable for concentrating biogenic amines and metabolites from biological fluids such as CSF that contain low concentrations of protein and other interfering substances. When concentrating compounds from plasma, which contains large quantities of protein and other electrochemically active materials, it is necessary to add an extraction step, such as alumina extraction. By substituting 0.05 M HCl for the conventional eluent, 0.1 M HClO4, we were able to increase recoveries of catecholamines from plasma by approximately 20%. Recovery of endogenous catecholamines from plasma following the combined alumina extraction - lyophilization procedure was 81 +/- 1%.     

A non-intrusive method for the measurement of water vapour sorption by bacterial spores

Single particle levitation (SPL) is used to measure the sorption and desorption of water vapour from microparticles comprising of Bacillus spores. Water gain is determined from increases in the weight-balancing levitation voltages. Spore water isotherms are compared to values previously reported using bulk gravimetric methods. Two salient differences are found between the SPL and bulk data: (1) greater osmotic swelling is observed in the SPL data; and (2) the SPL data exhibit open loop hysteresis while bulk data exhibit closed loop hysteresis.     

A simple method for the measurement of bacterial particle conductivities

A method was developed for the measurement of the bacterial particle conductivity, based on the measurement of the conductivity of a bacterial cell suspension sigma(s) and the suspending medium sigma(m). A line plotted through sigma(s) - sigma(m) versus sigma(m) crosses the x-axis at sigma(m) = sigma(p), independent of the bacterial cell concentration. The method does not require anything more complex than a centrifuge and a conductivity meter. Knowledge of the bacterial particle conductivity is of importance in, for example, the dielectrophoretic separation, manipulation and trapping of bacterial cells, as well as the study of their physiological state.     

The analysis of biogenic amines in the thoracic nervous system of the locust,Schistocerca gregaria,by gas-chromatography negative ion chemical ionization mass spectrometry (GC-NICIMS)

Biogenic amines in single ventral thoracic nerve cords of the locust, Schistocerca gregaria, have been identified and quantified by an extraction-derivatization procedure involving their reaction with ditrifluoromethylbenzoyl chloride (DTFMB) in the aqueous phase followed by extraction into an organic solvent, hydrolysis of phenolic esters and conversion of free hydroxyl groups to trimethylsilyl ethers. Subsequent analysis of these DTFMB-TMS derivatives by GC-NICIMS revealed that the molecular ion carried most (> 60%) of the ion current which made the method highly specific and gave a limit of detection below the picogram level. This publication establishes unequivocally that the principal amines in locust ventral thoracic nerve cord are p-tyramine, p-octopamine, dopamine and noradrenaline. 5-Hydroxytryptamine was determined using a previously published GC-NICIMS technique (Markey et al., Biomed. Mass Spectromet. 7, 301–304, 1981).     

Direct ELISA for estrone measurement in the feces of sows: prospects for rapid, sow-side pregnancy diagnosis

Both free and conjugated fecal estrogens were surveyed in sows by means of RIA's after extraction and column chromatography. Six different RIA's were performed using the same fecal suspension. Based on differences in concentration in feces from 6 pregnant and 4 nonpregnant sows, estrone (E1) was selected for the development of a homologous, competitive ELISA for pregnancy diagnosis purposes. Polyclonal antibodies were raised against 6-keto-E1-carboxymethyloxime coupled to BSA. Biotinylated E1 was used as the competitive agent and was detected with a streptavidin-peroxidase conjugate. Tetramethylbenzidine was used as chromogen. Validation of this direct, easy to perform ELISA showed satisfactory specificity, accuracy, precision and sensitivity. Comparison of the assay with a validated RIA (including extraction and column chromatography) resulted in a linear regression equation of ELISA = 0.88 RIA - 0.72 (r = 0.95; n = 24 fecal samples). The E1 values in feces from 11 pregnant sows during the first 40 d of gestation compared with values from 10 nonpregnant sows showed significant differences between Days 23 and 35 of pregnancy (P < 0.05). The highly significant difference in E1 concentrations (P < 0.0012) between Days 27 and 30 in particular offers promise for pregnancy diagnosis.     

A novel method for exploring elemental composition of microbial communities: Laser ablation-inductively coupled plasma-mass spectrometry of intact bacterial colonies

Bacterial colonies are spatially complex structures whose physiology is profoundly dependent on interactions between cells and with the underlying semi-solid substratum. Here, we use bacterial colonies as a model of a microbial community to evaluate the potential of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to delineate elemental distributions within colonies with minimal pre-treatment. To reduce water content of the colony and limit undesirable absorption of laser energy, we compared methods of preparing 24 h-old colonies of Escherichia coli TG1 on agar for laser ablation. Colonies on excised agar segments dried on chromatography paper were superior to colonies dried in a dessicator or by prolonged incubation, with respect to signal magnitude, signal:noise ratio and background signal. Having optimised laser scan speed (10 μm s^− 1) and laser beam diameter (100 μm), further improvements were achieved by growing colonies on nylon membranes over agar, which were then transferred to the ablation chamber without further treatment. Repeated line rasters across individual membrane-supported colonies yielded three-dimensional elemental maps of colonies, revealing a convex morphology consistent with visual inspection. By normalising isotope counts for P, Mn, Zn, Fe and Ca against Mg, the most abundant cellular divalent cation, we sought elemental heterogeneity within the colony. The normalised concentration of Mn in the perimeter was higher than in the colony interior, whereas the converse was true for Ca. LA-ICP-MS is a novel and powerful method for probing elemental composition and organisation within microbial communities and should find numerous applications in, for example, biofilm studies.     

A contamination reducing method by ion beam bombardment of the specimen in high resolution electron microscopy

The theory of contamination and a contamination reducing method are discussed on the basis of time dependent micrograph series and their tilted images for determining contamination.For a high current density of an electron probe in the field emission scanning electron microscope, it is observed that contaminated cones are formed in proportion to the exposure time of an electron beam. From the measurement of the contamination layer thickness and its area, the contamination rate and time dependent shape are formulated, mainly depending on the cross-section and current density together with the average lifetime of adsorption molecules.It is found that the contamination rate and stray contamination of outgassing molecules forming part of the specimen are effectively reduced by a pre-bombardment of argon ions on the surfaces of specimens. The contamination rate is reduced to a small extent (5%) using the present method.     

A rapid method for detecting bacterial contamination in the presence of Penicillium and Streptomyces antibiotic fermentations

Contamination in antibiotic fermentation processes results in major economic and process problems. The detection and elimination of contamination is a continual objective for fermentation companies. While efforts continue to eliminate contamination by improving equipment and sterile techniques, it is still imperative to have a rapid method for detecting contamination in laboratory-stage inoculum and seed tanks. This article describes the successful studies leading to the adoption of the BACTEC, an automatic bacterial detection system, as a supplemental detection technique. The BACTEC system detects contamination by incubating samples with a selected(14)C-labeled substrate or substrates. The resulting metabolism of substrate produces (14)C-labeled CO(2) which is then quantified and expressed as a growth index, permitting detection of contamination more rapidly at a much earlier time than is possible with conventional detection techniques that involve Phenol red dextrose broth, streak plates, and microscopic examination techniques.     

A method for measuring the internal pH in illuminated chloroplasts based on the stimulation of proton uptake by amines.

A simple method for measuring the internal pH of chloroplasts during steady-state illuminations based on the stimulation of proton uptake by monofunctional amines was developed. Predictions of a mathematical derivation concerning the dependence of the stimulation on the amine concentration, the internal volume, the pK of the amine and the external pH have been verified experimentally. To circumvent uncoupling and swelling due to large internal accumulation of amines extrapolation of the stimulation to low amine concentrations was suggested and shown to lead to valid values. Alternatively, swelling could be largely reduced in a medium containing potassium aspartate and valinomycin.     

Derivatization strategies for the determination of biogenic amines in wines by chromatographic and electrophoretic techniques

This paper revises the derivatization approaches for the determination of biogenic amines in wines. Since most of these amines display poor spectroscopic features to be detected by UV absorption or emission (fluorescence) spectroscopy, derivatization is necessary to attain the desired sensitivity. Reagents such as o-phthaldialdehyde, fluorenylmethylchloroformate, dansyl-Cl and dabsyl-Cl have widely been used for analytical labeling through amino group. A comparison of features of off- and on-line pre- and post chromatographic/electrophoretic labeling is given using 1,2-naphthoquinone-4-sulfonate (NQS) as an example. The evaluation of the influence of the wine sample composition on the derivatization process indicates that pre-column labeling may undergo more severe matrix effects.     

Uptake of biogenic amines by glial cells in culture I. A neuronal-like transport system for serotonin

Rat C6 astrocytoma cells take up serotonin (5HT) via a high affinity carrier mediated system with Km of 1 micromolar, and a second component of lower affinity. This high affinity 5HT transport system is rapid, concentrative, and highly sodium and temperature dependent. Chlorimipramine and Lilly 110140 preferentially block the glial 5HT but not NE uptake. This preferential inhibition has previously been shown for synaptosomes and brain slices. Norepinerphrine (NE) and to a lesser extent dopamine (DA) block the glial 5HT uptake, suggesting a partial overlap between the catecholamine and indoleamine glial carrier systems. 5-Hydroxy but not 6-hydroxy dopamine inhibits the high affinity 5HT transport in glia. A variety of ring hydroxylated indoleamine analogs block this glial 5HT transport; of the compounds tested, 5, 7 dihydroxytryptamine is the least effective inhibitor. Phenylethylamine (PEA) and its 0-methylated derivatives block synaptosomal and glial 5HT transport equally well. These observations suggest that cultured C6 cells used as models of glia possess a 5HT transport system which kinetically and pharmacologically resembles a neuronal 5HT transport system.