An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.     

Quantitation of Viral DNA by Real-Time PCR Applying Duplex Amplification, Internal Standardization, and Two-Color Fluorescence Detection

A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10² to at least 5 × 10⁶ copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.     

Comparison and evaluation of RNA quantification methods using viral, prokaryotic, and eukaryotic RNA over a 10 concentration range

Quantification of RNA is essential for various molecular biology studies. In this work, three quantification methods were evaluated: ultraviolet (UV) absorbance, microcapillary electrophoresis (MCE), and fluorescence-based quantification. Viral, bacterial, and eukaryotic RNA were measured in the 500 to 0.05-ng μl⁻¹ range via an ND-1000 spectrophotometer (UV), Agilent RNA 6000 kits (MCE), and Quant-iT RiboGreen assay (fluorescence). The precision and accuracy of each method were assessed and compared with a concentration derived independently using inductively coupled plasma-optical emission spectroscopy (ICP-OES). Cost, operator time and skill, and required sample volumes were also considered in the evaluation. Results indicate an ideal concentration range for each quantification technique to optimize accuracy and precision. The ND-1000 spectrophotometer exhibits high precision and accurately quantifies a 1-μl sample in the 500 to 5-ng μl⁻¹ range. The Quant-iT RiboGreen assay demonstrates high precision in the 1 to 0.05-ng μl⁻¹ range but is limited to lower RNA concentrations and is more costly than the ND-1000 spectrophotometer. The Agilent kits exhibit less precision than the ND-1000 spectrophotometer and Quant-iT RiboGreen assays in the 500 to 0.05-ng μl⁻¹ range. However, the Agilent kits require 1 μl of sample and can determine the integrity of the RNA, a useful feature for verifying whether the isolation process was successful.     

Spectrophotometric flow injection determination of caffeine in solid and slurry coffee and tea samples using supported liquid membranes

A method for the determination of caffeine in coffee and tea samples based on the use of supported liquid membranes coupled to a flow system has been developed. The sample may be analysed both as solid and slurry. In the case of solid sample, this is directly placed in the membrane unit, and when the sample is slurry, this is continuously pumped to the membrane unit. In both cases, the caffeine released from the sample passes through the membrane (PTFE/n-undecane:hexyl ether) into an acidic acceptor stream. This stream flows through a spectrophotometric detector allowing the measurement of the absorbance of caffeine at 274 nm. The method shows a linear range between 0.5 and 15 g l⁻¹, with a relative standard deviation of ±3.7% and a sample throughput of 7–8 samples h⁻¹.     

A method for separate quantitative determination of 2-keto-3-deoxy-octonate and 3,6-dideoxyhexoses in mixtures.

A method for analysing ³²P in small aqueous samples by measuring the ?erenkov radiation is described. Centering problems with small sample volumes are eliminated by placing the sample in a polystyrene tube in the scintillation vial. The detection efficiency is high, 54.5 ± 0.6% (2 SD) at a sample volume of 25 μl. The reproducibility is good and independent of the sample volume. The detection efficiency of ³²P in polyacrylamide gel is shown to be as good as in water.     

A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon

Zincon (2-carboxy-2′-hydroxy-5′-sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator’s versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn²⁺, Cu²⁺, and Co²⁺ were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, an optimized method that allows the facile determination of Zn²⁺, Cu²⁺, and Co²⁺ with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn²⁺ and Cu²⁺ within the same sample is described. Using the prototypical Cu²⁺/Zn²⁺-protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantification in metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins.     

Measuring protein synthesis using metabolic H labeling, high-resolution mass spectrometry, and an algorithm

We recently developed a method for estimating protein dynamics in vivo with heavy water (²H₂O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS) [16], and we confirmed that ²H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the ²H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT–ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given ²H₂O.     

Simple and rapid analytical method for detection of amino acids in blood using blood spot on filter paper, fast-GC/MS and isotope dilution technique

A simple and rapid method for quantitative analysis of amino acids, including valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and phenylalanine (Phe), in whole blood has been developed using GC/MS. In this method, whole blood was collected using a filter paper technique, and a 1/8 in. blood spot punch was used for sample preparation. Amino acids were extracted from the sample, and the extracts were purified using cation-exchange resins. The isotope dilution method using ²H₈-Val, ²H₃-Leu, ²H₃-Met and ²H₅-Phe as internal standards was applied. Following propyl chloroformate derivatization, the derivatives were analyzed using fast-GC/MS. The extraction recoveries using these techniques ranged from 69.8% to 87.9%, and analysis time for each sample was approximately 26 min. Calibration curves at concentrations from 0.0 to 1666.7 μmol/l for Val, Leu, Ile and Phe and from 0.0 to 333.3 μmol/l for Met showed good linearity with regression coefficients = 1. The method detection limits for Val, Leu, Ile, Met and Phe were 24.2, 16.7, 8.7, 1.5 and 12.9 μmol/l, respectively. This method was applied to blood spot samples obtained from patients with phenylketonuria (PKU), maple syrup urine disease (MSUD), hypermethionine and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and the analysis results showed that the concentrations of amino acids that characterize these diseases were increased. These results indicate that this method provides a simple and rapid procedure for precise determination of amino acids in whole blood.     

Determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid by high-performance liquid chromatography,using on-column concentration and electrochemical detection

Chromatographic conditions for the determination of 5-methyltetrahydrofolic acid in plasma and spinal fluid are described, involving simple pretreatment of the sample. Electrochemical detection was used. The linear range of the method is more than 10³. Recovery from plasma and spinal fluid is 100%, and the detection limit of the method is 2·10^?9M, sufficient for the detection of endogenous plasma and spinal fluid levels. The detection conditions are discussed. Endogenous concentrations of the compound in plasma and spinal fluid were determined and correlated with a folate bioassay. Plasma concentrations have been shown after the administration of leucovorin which is used in anticancer therapy.     

The Stone Brusher, a New Sampler for Submerged Epilithic Material in Shallow Streams and Lakes

The Stone Brusher is designed to take qualitative or semi-quantitative samples of material attached to stones at 7–50 cm depth in running or stagnant waters. The epilithic material is dislodged from the stone surface with a rotating brush enclosed in a chamber and the material is drawn up directly into the sample bottle with an air-cylinder. The operator takes a sample quickly and without putting hands into the water. The sampling area is about 28 cm². The sampler is made of plastic, stainless steel and aluminium and weighs 3.1 kg. The equipment is robust and easily handled and it is constructed to meet the demand for standardized sampling for research and environmental monitoring and to improve working conditions for sampling personnel. The equipment allows sampling from bedrock and large stones that cannot be lifted from the bottom and it can be used for reliable sampling also in fast-flowing streams where the dislodged material is easily flushed away. Using Near-Infrared Spectroscopy and diatom analyses, this new sampler is evaluated in comparison to the recognized toothbrush method, which indicates that the Stone Brusher reduces sampling variability compared with the toothbrush method.     

Simultaneous analysis of diphenylmethoxyacetic acid,a metabolite of diphenhydramine,and its deuterium-labeled stable isotope analog in ovine plasma and urine

Diphenylmethoxyacetic acid (DPMA) is a major metabolite of diphenhydramine in monkeys, dogs, and humans. The metabolic fate of diphenhydramine (DPHM) in sheep is not yet well understood; however, preliminary studies have demonstrated the presence of DPMA in the plasma and urine of sheep following an intravenous bolus of DPHM. Our current studies employ the simultaneous intravenous co-administration of DPHM and the stable isotope analog of DPHM to investigate the pharmacokinetics of DPHM in sheep. In these studies, in order to investigate the pharmacokinetics of the DPMA metabolite, measurement of both unlabeled and stable-isotope labeled DPMA is required. Thus, a stable isotope analog of DPMA ([²H₁₀]DPMA) was synthesized, characterized, and purified for use as an analytical standard. The quantitative method for the gas chromatography—electron-impact mass spectrometry (GC—EI-MS) analysis of DPMA and [²H₁₀]DPMA used a single step liquid-liquid extraction procedure using toluene for sample cleanup. The samples were derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide. A 1.0-μl aliquot of the prepared sample was injected into the GC-MS system and quantitated using selected-ion monitoring (SIM). One ion was monitored for each compound, namely, m/z 165 for the internal standard diphenylacetic acid, m/z 183 for DPMA, and m/z 177 for [²H₁₀]DPMA. The ion chromatograms were free from chromatographic peaks co-eluting with the compound of interest. The calibration curve was linear from 2.5 ng/ml (limit of quantitation) to 250.0 ng/ml in both urine and plasma. The intra-day and inter-day variabilities of this assay method were within acceptable limits (below 20% at the limit of quantitation and below 10% at all other concentrations). This method was used to measure the concentration of DPMA and [²H₁₀]DPMA in plasma and urine samples from a ewe in which equimolar amounts of DPHM and [²H₁₀]DPHM were administered by an intravenous bolus dose via the femoral vein. DPMA appeared to persist longer in the plasma and the urine as compared to DPHM. This method is robust and reliable for the quantitation of DPMA and [²H₁₀]DPMA in biological samples obtained from sheep (e.g. plasma and urine).     

The relation of sex,age, smoking status,birth rank and parental ages to pseudocholinesterase activity and phenotypes in a sample of Australian Caucasian adults

Summary The frequencies of the pseudocholinesterase alleles E₁ ^u, E₁ ^a and E₁ ^f have been determined in a random sample of Australian residents. The frequency of E₁ ^a is the highest yet reported in a large caucasian sample. The considerable variation in E₁ ^f frequencies in previously reported samples is discussed in terms of possible sources of error in fluoride number determinations.Enzyme activity was found to increase with age in adulthood and was higher in males than in females. It was also positively correlated with dibucaine number in type U subjects. These observations are in conflict with those reported in previous investigations.This work was supported by grants from the National Health and Medical Research Council of Australia and the Australian Tobacco Research Foundation.     

Investigation of the enzymatic activity of the Na,K-ATPase via isothermal titration microcalorimetry

Isothermal titration microcalorimetry (ITC) is shown here to be a sensitive and accurate method for assaying the steady-state enzyme activity of the Na⁺,K⁺-ATPase. Single ATP injection experiments yield an apparent enthalpy change for the ATP hydrolysis reaction catalyzed by the enzyme of −51 (± 1) kJ mol⁻¹. This value is independent of the amount of ADP accumulated in the sample cell, which indicates that under the experimental conditions studied here (saturating Na⁺ and K⁺ concentrations) ADP does not inhibit enzyme activity by reversal of the phosphorylation reaction and resynthesizing ATP. Multiple ATP injection titration experiments in which varying concentrations of ADP were initially included in the sample cell could be adequately explained by a Michaelis-Menten kinetic model incorporating noncompetitive inhibition. This suggests that ADP inhibits the enzyme by binding to more than one enzyme intermediate and inhibiting forward reactions of the enzyme. Values of K_m and K_I obtained for the fits agree with literature values obtained by other methods. Because ITC is a direct method of continually monitoring enzyme activity, it is a valuable supplement to less direct or noncontinuous methods such as colorimetric, enzyme-coupled or radioactivity-based assays.     

Individual closed chamber: an alternative method for quantifying 14C in both labeled organic and inorganic carbon substrates

Incubation of ¹⁴C-labeled substrates continues to be a widely used procedure in soil organic matter (OM) research due to its sensitiveness. When the labeling is found in liquid fractions (soil extracts, hydrolysates), ¹⁴C can be easily quantified by using an aliquot for scintillation counting. For this reason, converting a solid carbon sample into liquid form is a typical step for accurate ¹⁴C analysis. We have developed an alternative method to carry out this step, which uses standard glass hardware and does not require complex laboratory facilities. Carbon (both in organic or inorganic forms) is converted into CO₂ within a reaction vessel connected to a Twisselmann’s extractor with an alkali trap inside. This forms an individual closed chamber (ICC) for each sample, thus eliminating the risk of cross-contaminations. The alkali solution adsorbs the evolved CO₂ within the closed system, and the excess of pressure is easily overcome by the use of a balloon. We tested the procedure on a set of substrates and two contrasting soils, checking also the effect of different sample loads (from 20 to 160 mg C) on the CO₂ recovery of the process. The percentage of carbon recovered into the alkali (i.e. the efficiency of the process) ranged from 92% for the inorganic C to 93–95% for the organic C method, the latter being sensitive to the amount of sample used for analysis. The ICC method can be successfully applied to analyze ¹⁴C-labeling in both carbonates and OM from solid samples, thus representing an alternative method to some established protocols, and it is suitable for substrates with low or very low ¹⁴C contents, in which high volumes of sample must be analyzed in order to guarantee representative results.     

Plasma concentrations of temazepam,a 3-hydroxy benzodiazepine,determined by electron-capture gas—liquid chromatography

A simple and sensitive high-performance liquid chromatographic assay of methotrexate (MTX) and its two active metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-di-amino-N¹⁰-methylpteroic acid (APA) in plasma, saliva and urine was developed. The method involved deproteinization with acetonitrile followed by addition of isoamyl alcohol and ethyl acetate. After extraction the sample was chromatographed on a cation-exchange column and monitored at 313 nm. The retention times were 5, 7 and 9 min and detection limits 20, 10 and 5 ng/ml for 7-OH-MTX, MTX and APA, respectively. For concentrations greater than 100 ng/ml one-step deproteinization of 0.1 ml sample with 0.25 ml acetonitrile was satisfactory for sample preparation. The method has been evaluated in samples from patients and rabbits receiving MTX.     

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.^1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.⁶ As every chemical vibration is assigned to a specific Raman band (wavenumber in cm^-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.^7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.¹ Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).¹⁰Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO₂) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.     

Hemoglobins, haptoglobins, and transferrins in indigenous populations of Kenya

Hemoglobin, haptoglobin, and transferrin phenotypes were determined by means of starch-gel electrophoresis for a sample of 226 Kenya hospital patients. Allele frequencies were: Hb_β^S=0.081; Hp¹=0.057; Tf^D=0.038. Hemoglobin S was the only aberrant hemoglobin found in this sample. Transferrins C and D were the only transferrins found. Hemoglobin and transferrin phenotypes were also determined for a sample of 201 newborn Kenya infants. One of these infants had hemoglobins F, S, and C, eight had hemoglobins A, F, and S, and the remainder had hemoglobins A and F. Transferrins C, B, and D were found in this sample. Allele frequencies were: Tf^B=0.008; Tf^D=0.019.     

A rapid microtechnique for the preparation of biological material for the simultaneous analysis of calcium,magnesium and protein

No simple documented method of sample preparation is available for the analysis of calcium and magnesium in biological samples despite increasing awareness of the biological roles of these cations. The technique described here is rapid, avoids the use of dangerous reagents or costly equipment, and allows accurate determination of protein, calcium, and magnesium content of the speciment after sample preparation. Tissue is solubilized in 1 n NaOH after which one aliquot is used for protein analysis by the method of Lowry et al. (1951) (J. Biol. Chem.193, 265–275), and another is used for determination of cations by atomic absorption spectroscopy. The method was used to analyze biological samples including adipocyte subcellular fractions, bovine liver, and orchard leaves. Results correlated well with those using reference wet ashing and low-temperature ashing techniques with correlation coefficients (r²) of 0.95 for calcium and 0.997 for magnesium. Intraassay coefficients of variation were 4–4.2%. The base-digestion technique is a simple, rapid, and precise method which avoids most of the limitations of currently available sample preparation techniques.     

Method for simultaneous measurement of total and radioactive carbon in soils,soil extracts and plant materials

Summary A rapid procedure is proposed for simultaneous measurement of total and radioactive carbon in soils, soil extracts and plant materials. The procedure involves dry or wet combustion of the sample, total carbon determination with an automatic analyser and C¹⁴O₂ absorbtion in a liquid for scintillation measurement. The use of methyl-cellosolve plus mono-ethanolamine as a CO₂ absorber allows measurements of weakly labelled materials. This method is suitable for fast routine analysis. re]19750929