Semiautomatic photometric screening of urine samples for significant bacteriuria with high predictive values.

A simple method for screening urine specimens for significant gramnegative bacteriuria is presented, in which microtitration plates, a vertical-beam photometer used for reading ELISA and an inexpensive microcomputer are employed. The wells of the plate containing brain-heart infusion broth are inoculated with urine. Every hour turbidity is measured and the values are compared with the original ones. The whole examination is terminated in five hours. At the actual prevalence of 0.27, specificity is 0.97, the predictive value of a negative result is 0.97 and false positives 0.03. The method is used for rapid reporting to the ward, and for subsequent differentiated standard cultivation.     

Quantitative assays for uracil-DNA glycosylase of high sensitivity

We have developed a sensitive fluorometric assay using bisulfite deaminated (C----U), covalently-closed circular PM2 DNA as the substrate. We describe a reliable way to prepare this substrate without nicking the PM2 DNA. Methods, which depend on toluenization of the cells, are described for reproducibly and quantitatively assaying uracil-DNA glycosylase. The sensitivity is such that only 200 EL4 mouse thymoma cells or 30,000 Escherichia coli cells are needed for each point in a kinetic assay.     

An automated continuous determination of oxygen with high sensitivity

A sensitive, rapid, and precise method is described for the continuous determination of oxygen in gases. The principle of the method is based on the reaction of O₂ with an alkaline catechol or pyrogallol solution, which is combined with a Fe²⁺ solution to increase the sensitivity of the color reaction. The development of the color takes place in a tube system provided with a proportioning pump and is read automatically on a recorder after passing a flow cell of a photometer. The lower limit of sensitivity of this method is 0.05 μl of O₂ min^?1. Thus, in a gas flow of ≈0.5 ml min^?1, an oxygen concentration of ≥0.01% (v/v) can be determined. If the gas flow is increased to ≈2.5 ml min^?1, this limit of sensitivity is lowered to ≥0.002% (v/v). Since a 2-min period is necessary for the measurement, the volume of the sample has to be 1 ml in the first case and 5 ml in the second.     

Simple photometric and fluorometric assays are insufficient for assessing UV-induced mutagenesis in genes controlling tryptophan synthesis in Escherichia coli

A new approach to detecting induced mutations was tested based on the assay of cell extracts and special growth media following cultivation of UV irradiated Escherichia coli cells. No correlation was found between the UV dose and the optical densities of cultural media or cell extracts prepared by Triton X-100 treatment. Blue fluorescence of concentrated cultural media varied with cell dose, according to a rather complex law, which differed substantially from the known dose-effect curves for induced mutations. Nevertheless, a certain extent of the brown staining of tryptophan containing medium could, presumably, serve as a quite sensitive indicator of the integral metabolic activity of bacteria grown in the medium. Besides, we observed that overnight lag phase cultures became gradually more transparent, when analysed in the spectrophotometer cuvette just after their dilution with fresh medium.     

A novel microgravimetric DNA sensor with high sensitivity

A novel method using an amplifier with a cantilever and gold nanoparticles successfully to extend the length of the target for the specific and high sensitive detection of DNA was reported. When the size of gold nanoparticle is 50 nm, a sensitivity of 10(-15)M for the single base mutation detection has been achieved.     

A high sensitivity system for luminescence measurement of materials

A unique combined and multi‐disciplinary wavelength multiplexed spectrometer is described. It is furnished with high‐sensitivity imaging plate detectors, the power to which can be gated to provide time‐resolved data. The system is capable of collecting spectrally resolved luminescence data following X‐ray excitation [radioluminescence (RL) or X‐ray excited optical luminescence (XEOL)], electron irradiation [cathodoluminescence (CL)] and visible light from light emitting diodes (LEDs) [photoluminescence (PL)]. Time‐resolved PL and CL data can be collected to provide lifetime estimates with half‐lives from microsecond timeframes. There are temperature stages for the high and low temperature experiments providing temperature control from 20 to 673 K. Combining irradiation, time resolved (TR) and TR‐PL allows spectrally‐resolved thermoluminescence (TL) and optically stimulated luminescence (OSL). The design of two detectors with matched gratings gives optimum sensitivity for the system. Examples which show the advantages and multi‐use of the spectrometer are listed. Potential future experiments involving lifetime analysis as a function of irradiation, dose and temperature plus pump‐probe experiments are discussed.     

A new approach to high sensitivity differential hybridization

We describe a new approach to differential hybridization, designed to identify cDNA clones representing rare mRNA species. Duplicate filters carrying a library of cDNA from phorbolmyristate acetate (PMA)-induced EL-4 cells in λgt11 were hybridized with high concentrations of unlabeled, cloned, single-stranded cDNA from induced and control EL-4 cells, respectively. Plaques binding single-stranded cDNA were revealed by a second round of hybridization with ³⁵S-labeled DNA complementary to the vector moiety of the single-stranded cDNA. Plaques corresponding to PMA-induced mRNAs occurring at a level of about 1 part in 15000 were isolated. We believe the method is at least ten times more sensitive than conventional differential hybridization.     

Capillary electrophoresis of chitooligosaccharides in acidic solution: Simple determination using a quaternary-ammonium-modified column and indirect photometric detection with Crystal Violet

Five chitosan oligosaccharides were separated in acidic aqueous solution by capillary electrophoresis (CE) with indirect photometric detection using a positively coated capillary. Electrophoretic mobility of the chitooligosaccharides (COSs) depended on the number of monomer units in acidic aqueous solution, similar to other polyelectrolyte oligomers. The separation was developed in nitric acid aqueous solution at pH 3.0 with 1 mM Crystal Violet, using a capillary positively coated with N-trimethoxypropyl-N,N,N-trimethylammonium chloride. The limit of the detection for chitooligosaccharides with two to six saccharide chains was less than 5 μM. CE determination of an enzymatically hydrolyzed COS agreed with results from HPLC.     

Resolution and sensitivity of high field nuclear magnetic resonance spectroscopy

NMR frequency assignments are usually considered a prerequisite for the analysis of NOESY spectra, in turn required for the calculation of biomolecular structures. In contrast, as we propose here, relatively high numbers of unambiguous NOE identities can be consistently achieved in an automated manner by relying only on grouping resonances into connected spin systems. To achieve this goal, we have developed for proteins two protocols, SPI and BACUS, based on Bayesian inference. SPI (Grishaev and Llinás, 2002c) produces a list of the (1)H resonance frequencies from homo- and hetero-nuclear multidimensional spectra, grouped into effective spin systems. BACUS automatically establishes probabilistic identities of NOESY cross-peaks in terms of the chemical shifts provided by SPI. BACUS requires neither assignment of resonances nor an initial structural model. It successfully copes with chemical shift overlap and does so without cycling through 3D structure calculations. The method exploits the self-consistency of the NOESY graph by taking advantage of a network of J- as well as NOE-connected "reporter" protons sorted via SPI. BACUS was validated by tests on experimental NOESY data recorded for the col 2 and kringle 2 domains.     

Covalent attachment of peptides for high sensitivity solid-phase sequence analysis

We have developed a method for the high efficiency covalent immobilization of picomole to nanomole quantities of peptides in a form compatible with high sensitivity gas-liquid or solid-phase sequence analysis. Glass fiber filter paper was derivatized with amino-phenyltriethoxysilane and peptides were applied to circular disks cut to 1-cm diameters. Peptides were covalently immobilized on the aminophenyl-glass fiber paper through their terminal alpha-carboxyl groups and amino acid side-chain carboxyl groups by activation with the water-soluble reagent N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide. Disks containing the covalently attached peptide were directly inserted into the cartridge of an automated sequenator for sequence analysis by the Edman degradation. Peptides prepared in this way could be routinely sequenced through to and including the C-terminal amino acid residue, at extraordinarily low backgrounds. The covalent immobilization of peptide fragments allowed far more flexibility in sequencing conditions, including the use of polar extraction solvents to increase the yield of phenylthiohydantoin (PTH)-His and PTH-Arg and the use of alternative Edman-type sequencing reagents with enhanced detectability, such as the chromophoric compound 4- (N,N'-dimethylamino)azobenzene-4'-isothiocyanate. The potential of this high efficiency immobilization method for contributing to the development of sequencing chemistries with enhanced sensitivity is discussed.     

Goldfish color vision sensitivity is high under light-adapted conditions

The wavelength discrimination threshold of three goldfish was examined in a series of behavioral experiments. Using an auto-shaping technique, detection thresholds were established for 531 and 648 nm spectral increments presented on a 6.6 cd m^–2 white background. Next, discrimination between the wavelengths was established at equal, suprathreshold, intensities. Finally, the intensities of the two stimuli were reduced to establish the intensity threshold for the wavelength discrimination. The results indicate that goldfish, like several mammalian species, can discriminate wavelength at detection threshold intensity. This finding suggests that high color sensitivity is not confined to mammals or dependent upon a very high percentage of wavelength opponent ganglion cells. Rather, high color vision sensitivity may be based upon an inherent sensitivity advantage of wavelength opponent receptive fields compared to non-wavelength opponent receptive fields and be an important selective advantage of wavelength opponency and color vision.