A simple imaging method for biomass determination

An inexpensive and fast method based on images taken during growth of bacterial cells on multi-well plates was developed for biomass quantification. A correlation of 85% between the results obtained by image analysis and optical density measurements was obtained. This simple method allows the assessment of growth with highly aggregated cell cultures and the rapid screening of a large number of carbon sources.     

A simple spectrophotometric assay for arogenate dehydratase

A simple spectrophotometric assay for arogenate dehydratase, the enzyme that catalyzes the formation of L-phenylalanine from L-arogenate, is presented. The method couples the arogenate dehydratase reaction with that of an aromatic aminotransferase partially purified from Acinetobacter calcoaceticus. In the presence of 2-ketoglutarate, phenylpyruvate formation is measured at 320 nm at basic pH. The method was compared with two other methods already in use in our laboratory for arogenate dehydratase. The new method is simple, quick, fairly sensitive, and especially suitable for the screening of a large number of samples.     

A simple radiochemical assay for prostaglandin synthetase

A simple radiochemical procedure for prostaglandin synthetase is described, in which incubation mixtures are applied to small disposable columns of Bio-Sil A (silica gel) resin and eluted with two different solvent mixtures to achieve a separation of unmetabolized substrates from prostaglandin products.     

A simple micro-growth assay for enumerating bacteria

A simple method for nonspecific determination of bacteria concentrations in a variety of liquid samples was developed. The assay was based on the time required for a sample grown in liquid media to reach a threshold turbidity. Samples were combined with media in a covered 96-well microwell plate and the turbidity was monitored in real time as the bacteria grew in a temperature-controlled plate reader. A significant problem with growth in microwells was condensation on the cover, which prevented accurate turbidity measurement. This problem was overcome by coating the cover with a small amount of surface-active agent. Salmonella and E. coli concentrations could be determined with a relative error of approximately 20% at levels from 10 to 10(6) cells/ml (eight replicates). An assay of 10 samples with standards required 10 min to set up and 20 min for data processing using a computer spreadsheet program. Growth time at 37 degrees C ranged from 4 h for samples at 10(7) cells/ml to 16 h for samples at 10 cells/ml.     

A simple fluorimetric assay for guanosine nucleotides

It is comparatively easy to assay adenosine 5′-triphosphate either in the presence or absence of other nucleoside triphosphates (1). An assay for UTP has been reported (2) based on its incorporation into RNA using RNA polymerase and a poly(dA-dT) template in the presence of excess [³H]-ATP; this method could be adapted to assay for GTP and CTP. It is known that the fluorescence of Tb³⁺ increases on binding to nucleic acids (3–5), and this enhancement has been attributed both to complex formation between Tb³⁺ and guanine residues in the nucleic acid (3) and also to binding of Tb³⁺ to thiouracil (4).     

A simple enzyme-linked immunosorbent assay for testosterone

A new, enzyme-linked, immunosorbent assay for testosterone is described. The assay uses horseradish peroxidase coupled to testosterone as the tracer and offers the same sensitivity and reliability but greater convenience than radioimmunoassay. The assay is also simpler and more rapidly completed than previously described assays for testosterone.     

A simple assay procedure for beta-D-mannanase.

A simple assay procedure for beta-D-mannanase enzyme has been developed which employs carob D-galacto-D-mannan dyed with Remazolbrilliant Blue. Additionally, the procedure is quantitative, relatively sensitive, and highly specific for beta-D-mannanase enzyme. It can be readily used for the determination of beta-D-mannanase activity in crude enzyme preparations and column-chromatography eluates.     

A simple and sensitive assay for dopamine-beta-hydroxylase

A simple, rapid, and highly sensitive radiochemical assay for measuring the activity of dopamine-β-hydroxylase in tissues and serum is described. Enzyme activity is detected by converting [1-¹⁴C]tyramine to [1-¹⁴C]octopamine which is then subjected to periodate cleavage to form [¹⁴C]form-amide. This radiolabeled product is oxidized to ¹⁴CO₂ by addition of permanganate and the ¹⁴CO₂ is trapped and counted. The assay is simple and sensitive, it can linearly detect enzyme in all tissues with a wide range of activity, it uses maximal concentration of substrate, and it requires the addition of only one concentration of EMI to block endogenous inhibitor(s) in different tissues or enzyme concentrations.     

A simple activity assay for thrombin and hirudin

Cloning of the thrombin cDNA has made it possible to study thrombin function by site-directed mutagenesis. Quantitative results from studies of thrombin mutants are often hindered by difficulties in assaying the enzyme activity. The high enzyme concentrations required for activity determination by standard methods limit their usefulness to thrombin mutants that cannot be readily produced in large quantities. We have developed a novel method using the synthetic substrate S-2238 and hirudin, a tight-binding inhibitor of thrombin, that allows for the active-site titration of thrombin at concentrations as low as 20 pM, with an error of 5%. In addition, hirudin activity can be determined by this method to concentrations as low as 40 pM, with an error of 5%.     

A simple and sensitive radioreceptor assay for leukotrienes

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [3H]leukotriene D4 (LTD4) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD4. The values for fifty percent inhibition of bound [3H]LTD4 was 1.5 nM for LTD4, 45 nM for LTC4 and 24 nM for LTE4. LTB4 at 3.0 X 10(-5)M had no effect on [3H]LTD4 binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC4 and LTD4. However, in the presence of 20 mM serine-borate this method was highly specific for LTD4. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD4. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient 'r' of 0.992. The assay was also validated by quantitation of Lts released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD4 than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD4 specifically or LTC4 plus LTD4 simultaneously in one cell system.