A rapid and sensitive method for measuring ribonucleic acid in ribosomal preparations.

By using the fluorescence enhancement of ethidium bromide when it binds to RNA, a very rapid, simple and sensitive assay for the concentration of ribosomal RNA in complex mixtures has been devised.     

A sensitive method for measuring oxygen consumption

A technique is presented whereby oxygen consumption rates of the order of 10⁻⁶ μl/hr can be measured, thus providing a means for studying the respiration rates of single cells, even slowly respiring ones. The technique is based on the principle of incubating cells in extremely small chambers containing highly concentrated hemoglobin solutions. The absorbance shift occurring when hemoglobin is transformed from its oxygenated to its deoxygenated form is recorded microspectrophotometrically. The results obtained by this technique seem to be well in accordance with those of the Warburg manometric method. The technique is convenient and easy to handle and the sensitivity can be varied within wide limits, permitting different types of materials to be studied. In experiments using yeast cells, respiration rates from 1.0 × 10⁻⁶ to 1.8 × 10⁻⁶ μl O₂/cell/hr were revealed.     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

A rapid and sensitive method for the analysis of cyanophycin

A method has been devised for the quantitative analysis of cyanophycin, based on (1)H nuclear magnetic resonance (NMR) spectroscopy, allowing determination of the nitrogen status of cyanobacteria. Cyanophycin is extracted with minimal washing from small volumes of cells and quantified by integration of the NMR peak attributed to the protons attached to the delta-carbon of arginine. Linear relationships were found between the amount of cyanophycin determined by this method and both known concentrations of cyanophycin solutions and the amount of cyanophycin determined using the standard chemical arginine assay.     

A rapid and sensitive method for measuring the conditional emotional response. II: On-the-baseline excitatory conditioning and extinction

The present study outlines a rapid and sensitive on-the-baseline conditional emotional response (CER) procedure. Using rats as the experimental subject, the method detects delay conditioning, incubation, extinction and spontaneous recovery. In addition, the method detects conditional responding using electric shock ranging from 0.23 to 0.50 mA as the unconditional stimulus. Because of its speed and sensitivity, the method shelters the subject from unnecessary long-term deprivation and pain.     

A rapid method for measuring intracellular pH using BCECF-AM

A rapid intracellular pH (pH(i)) measurement method based on initial rate of increase of fluorescence ratio of 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein upon dye addition to a cell suspension in growth medium is reported. A dye transport model that describes dye concentration and fluorescence values in intracellular and extracellular spaces provides the mathematical basis for the approach. Experimental results of ammonium chloride challenge response of the two suspension cells, Spodoptera frugiperda and Chinese hamster ovary (CHO) cells, successfully compared with results obtained using traditional perfusion method. Since the cell suspension does not require any preparation, measurement of pH(i) can be completed in about 1 min minimizing any potential errors due to dye leakage.     

A new rapid fluorogenic method for measuring bacteriocin activity

We describe a novel bacteriocin screening assay based on fluorescence emitted by berberine following its influx into compromised cells. This technique showed agreement with the conventional well-diffusion method, and results can be obtained within one hour. This assay could facilitate the rapid identification of bacteriocinogenic bacterial isolates.     

A method for rapid freeze fixation of plant cells

Summary We describe here an apparatus that permits rapid freeze fixation of whole cells, which are then prepared by freeze substitution and resin embedment for examination in the EM. The freezing device utilizes a rotary solenoid that rapidly plunges the specimen holder, a formvar-film-covered thin wire loop, into a well of stirred liquid propane at –180C. The rotary solenoid allows for an adjustable, repeatable immersion rate. Substitution takes place at –80 C in acetone with 2% OsO₄ and is followed by en bloc staining in either hafnium tetrachloride or uranyl acetate. We have utilized these techniques on plant cells, for which there has been relatively little published work when compared to other organisms. The results show that, with the versatile specimen holder and rapid, repeatable immersion rates, different cell types, including pollen, stamen hairs, and germinating moss spores, can be rapidly frozen with repeatable success. The improved preservation achieved with rapid freeze fixation over conventional chemical fixation reveals itself particularly in the structure of the plasmamembrane, the cytoskeleton, chromatin, and certain endomembrane systems.     

A rapid and sensitive method for the detection of histone peptides

Fluorescamine has been used to obtain a peptide map of a mixture of histones (H₃, H₂A, H₂B, and H₄) prepared from oocytes of Xenopus laevis. Fluorescamine was found to be more sensitive than o-phthalaldehyde or ninhydrin-Cd for the detection of peptide fragments obtained from tryptic digestion of oocyte histones of X. laevis and the peptic digestion of the β chain of insulin. Using the β chain of insulin for a comparison, the 8 major peptide fragments could be separated by electrophoresis within 30 min and were detectable at the picomols level. Some 70 peptide spots of X. laevis oocyte histones were resolved, thus permitting the analysis of this complex mixture of polypeptides without the need for prior separation.     

A rapid sampling method for measuring efflux from erythrocytes.

A method is described for obtaining kinetic data using a water-jacketed Hamilton gas-tight syringe as a reaction vessel and delivering aliquots of the reaction mixture to a quench solution at intervals as small as 2 sec apart by means of a repeating dispenser attachment. This apparatus has been used to measure the rate of [¹⁴C]uridine equilibrium exchange efflux from human erythrocytes at 15°C and at uridine concentrations near the K_m for the transport process. It should be useful for kinetic studies of any reaction having a half time of the order of 4 sec or more, provided a method of rapidly quenching the reaction is available.     

A rapid bioluminescence method for quantifying bacterial adhesion to polystyrene

Bioluminescence ATP analysis has been used to assess bacterial adhesion with hydrophobic polystyrene tubes as the attachment surface. The assay was performed at 37 degrees C and pH 6.8 with a 10 min incubation period. A variation of more than 200-fold was observed in the adherence capacity of 34 urinary isolates of Escherichia coli, and organisms could be classified as strongly or weakly adherent. All strains capable of strong adhesion possessed both type 1 fimbriae and flagella, and maximum adhesion was expressed during the exponential growth phase. Attachment was in all cases virtually eliminated by addition of 2.5% (w/v) D-mannose to the incubation buffer. Conversely, strains which were deficient in type 1 fimbriae or flagella, or both, were weakly adherent during all phases of growth. There was no correlation between adherence of E. coli to polystyrene and adherence to buccal or uroepithelial cells, but there was a significant association with adherence to uromucoid (P less than 0.002).     

A rapid and sensitive method for the screening of DNA intercalating antibiotics

An improved, rapid and inexpensive gel mobility shift assay was developed for the screening of anthracycline antibiotics. The assay based on the intercalation activity of these molecules into dsDNA was used to assess the activity of partially purified antibiotics. Detection limits were of 0.1 ng ml^–1 with an average run time of 2 h. The assay is potentially useful for high throughput screening in bioprospecting, for monitoring fermentation production phases and downstream purification process.