Methods for extracting genomic DNA from whole blood samples : current perspectives

Deoxyribonucleic acid (DNA) extraction has considerably evolved since it was initially performed back in 1869. It is the first step required for many of the available downstream applications used in the field of molecular biology. Whole blood samples are one of the main sources used to obtain DNA, and there are many different protocols available to perform nucleic acid extraction on such samples. These methods vary from very basic manual protocols to more sophisticated methods included in automated DNA extraction protocols. Based on the wide range of available options, it would be ideal to determine the ones that perform best in terms of cost-effectiveness and time efficiency. We have reviewed DNA extraction history and the most commonly used methods for DNA extraction from whole blood samples, highlighting their individual advantages and disadvantages. We also searched current scientific literature to find studies comparing different nucleic acid extraction methods, to determine the best available choice. Based on our research, we have determined that there is not enough scientific evidence to support one particular DNA extraction method from whole blood samples. Choosing a suitable method is still a process that requires consideration of many different factors, and more research is needed to validate choices made at facilities around the world.

Critical review on the stability of illicit drugs in sewers and wastewater samples

Wastewater-based epidemiology (WBE) applies advanced analytical methods to quantify drug residues in wastewater with the aim to estimate illicit drug use at the population level. Transformation processes during transport in sewers (chemical and biological reactors) and storage of wastewater samples before analysis are expected to change concentrations of different drugs to varying degrees. Ignoring transformation for drugs with low to medium stability will lead to an unknown degree of systematic under- or overestimation of drug use, which should be avoided. This review aims to summarize the current knowledge related to the stability of commonly investigated drugs and, furthermore, suggest a more effective approach to future experiments. From over 100 WBE studies, around 50 mentioned the importance of stability and 24 included tests in wastewater. Most focused on in-sample stability (i.e., sample preparation, preservation and storage) and some extrapolated to in-sewer stability (i.e., during transport in real sewers). While consistent results were reported for rather stable compounds (e.g., MDMA and methamphetamine), a varying range of stability under different or similar conditions was observed for other compounds (e.g., cocaine, amphetamine and morphine). Wastewater composition can vary considerably over time, and different conditions prevail in different sewer systems. In summary, this indicates that more systematic studies are needed to: i) cover the range of possible conditions in sewers and ii) compare results more objectively. To facilitate the latter, we propose a set of parameters that should be reported for in-sewer stability experiments. Finally, a best practice of sample collection, preservation, and preparation before analysis is suggested in order to minimize transformation during these steps.

Estimation of elasticity map of soft biological tissue mimicking phantom using laser speckle contrast analysis

Scattering of coherent light from scattering particles causes phase shift to the scattered light. The interference of unscattered and scattered light causes the formation of speckles. When the scattering particles, under the influence of an ultrasound (US) pressure wave, vibrate, the phase shift fluctuates, thereby causing fluctuation in speckle intensity. We use the laser speckle contrast analysis (LSCA) to reconstruct a map of the elastic property (Young's modulus) of soft tissue-mimicking phantom. The displacement of the scatters is inversely related to the Young's modulus of the medium. The elastic properties of soft biological tissues vary, many fold with malignancy. The experimental results show that laser speckle contrast (LSC) is very sensitive to the pathological changes in a soft tissue medium. The experiments are carried out on a phantom with two cylindrical inclusions of sizes 6 mm in diameter, separated by 8 mm between them. Three samples are made. One inclusion has Young's modulus E of 40 kPa. The second inclusion has either a Young's modulus E of 20 kPa, or scattering coefficient of mu'(s), = 3.00 mm(-1) or absorption coefficient of mu(a) = 0.03 mm(-1). The optical absorption (mu(a)), reduced scattering (mu'(s)) coefficient, and the Young's modulus of the background are mu(a) = 0.01 mm(-1), mu'(s) = 1.00 mm(-1) and 12kPa, respectively. The experiments are carried out on all three phantoms. On a phantom with two inclusions of Young's modulus of 20 and 40 kPa, the measured relative speckle image contrasts are 36.55% and 63.72%, respectively. Experiments are repeated on phantoms with inclusions of mu(a) = 0.03 mm-1, E = 40 kPa and mu'(s) = 3.00 mm(-1). The results show that it is possible to detect inclusions with contrasts in optical absorption, optical scattering, and Young's modulus. Studies of the variation of laser speckle contrast with ultrasound driving force for various values of mu(a), mu'(s), and Young's modulus of the tissue mimicking medium are also carried out. (C) 2011 American Institute of Physics. doi:10.1063/1.3592352]

Bright-field and fluorescence chip-scale microscopy for biological imaging

Optical microscopy is an essential tool in biological science and one of the gold standards for medical examinations. Miniaturization of microscopes can be a crucial stepping stone towards realizing compact, cost-effective and portable platforms for biomedical research and healthcare. This thesis reports on implementations of bright-field and fluorescence chip-scale microscopes for a variety of biological imaging applications. The term “chip-scale microscopy” refers to lensless imaging techniques realized in the form of mass-producible semiconductor devices, which transforms the fundamental design of optical microscopes.

Our strategy for chip-scale microscopy involves utilization of low-cost Complementary metal Oxide Semiconductor (CMOS) image sensors, computational image processing and micro-fabricated structural components. First, the sub-pixel resolving optofluidic microscope (SROFM), will be presented, which combines microfluidics and pixel super-resolution image reconstruction to perform high-throughput imaging of fluidic samples, such as blood cells. We discuss design parameters and construction of the device, as well as the resulting images and the resolution of the device, which was 0.66 µm at the highest acuity. The potential applications of SROFM for clinical diagnosis of malaria in the resource-limited settings is discussed.

Next, the implementations of ePetri, a self-imaging Petri dish platform with microscopy resolution, are presented. Here, we simply place the sample of interest on the surface of the image sensor and capture the direct shadow images under the illumination. By taking advantage of the inherent motion of the microorganisms, we achieve high resolution (~1 µm) imaging and long term culture of motile microorganisms over ultra large field-of-view (5.7 mm × 4.4 mm) in a specialized ePetri platform. We apply the pixel super-resolution reconstruction to a set of low-resolution shadow images of the microorganisms as they move across the sensing area of an image sensor chip and render an improved resolution image. We perform longitudinal study of Euglena gracilis cultured in an ePetri platform and image based analysis on the motion and morphology of the cells. The ePetri device for imaging non-motile cells are also demonstrated, by using the sweeping illumination of a light emitting diode (LED) matrix for pixel super-resolution reconstruction of sub-pixel shifted shadow images. Using this prototype device, we demonstrate the detection of waterborne parasites for the effective diagnosis of enteric parasite infection in resource-limited settings.

Then, we demonstrate the adaptation of a smartphone’s camera to function as a compact lensless microscope, which uses ambient illumination as its light source and does not require the incorporation of a dedicated light source. The method is also based on the image reconstruction with sweeping illumination technique, where the sequence of images are captured while the user is manually tilting the device around any ambient light source, such as the sun or a lamp. Image acquisition and reconstruction is performed on the device using a custom-built android application, constructing a stand-alone imaging device for field applications. We discuss the construction of the device using a commercial smartphone and demonstrate the imaging capabilities of our system.

Finally, we report on the implementation of fluorescence chip-scale microscope, based on a silo-filter structure fabricated on the pixel array of a CMOS image sensor. The extruded pixel design with metal walls between neighboring pixels successfully guides fluorescence emission through the thick absorptive filter to the photodiode layer of a pixel. Our silo-filter CMOS image sensor prototype achieves 13-µm resolution for fluorescence imaging over a wide field-of-view (4.8 mm × 4.4 mm). Here, we demonstrate bright-field and fluorescence longitudinal imaging of living cells in a compact, low-cost configuration.

A simple method for counting bacteria with active electron transport system in water and sediment samples [Translation from: Kiel. Meeresforsch. 31(2) 83-86, 1975]

Description of a simple method for counting bacteria with active electron transport systems in water and sediment samples. Sodium succinate, NADH and NADPH served as electron donors. It is possible to see several sites of electron transport in the larger cells. Especially impressive are the plankton-algae, protozoa, and small metazoa. This is a partial translation of the ”method” section only.

Comparison of systems of biological indication approved during the course of the joint Anglo-Soviet investigations held under the auspices of the Institute of Hydrobiology of the Academy of Sciences of the Ukrainian SSR

In accordance with the plan for joint Anglo-Soviet scientific and technical collaboration on environmental problems, the comparative evaluation of systems of hydrobiological analysis of the surface water quality started in 1977 at the Regional Laboratory of the Severn-Trent Water Authority in Nottingham were continued in the spring of 1978. The investigations were carried out under the auspices of the Institute of Hydrobiology of the Academy of Sciences of the Ukrainian SSR. Hydrobiological and hydrochemical samples were collected by Soviet and British specialists from the Kiev reservoir and the rivers Dnieper, Sozh, Desna and Snov. The samples were processed on the expedition ships and in the Laboratory for the Hydrobiology of Small Water Bodies of the Institute of Hydrobiology of the Academy of Sciences of the Ukrainian SSR. The possible approved methods to be adopted were evaluated from the samples using the phytoperiphyton, phytoplankton, zooplankton and zoobenthos against a background of hydrochemical characteristics. The study concludes that weather conditions complicated the work on testing the systems of biological indication of water quality and made it inadvisable to use those methods of comparison which were used when similar work was carried out in Nottingham.

Approval of systems of Biological Indication of Water Quality under the auspices of the Severn-Trent Water Authority, UK

In accordance with the plan for joint Anglo-Soviet scientific and technical collaboration on environmental problems, the comparative evaluation of systems of hydrobiological analysis of the surface water quality started in 1977 at the Regional Laboratory of the Severn-Trent Water Authority in Nottingham were continued in the spring of 1978. The investigations were carried out under the auspices of the Institute of Hydrobiology of the Academy of Sciences of the Ukrainian SSR. Hydrobiological and hydrochemical samples were collected by Soviet and British specialists from the Kiev reservoir and the rivers Dnieper, Sozh, Desna and Snov. The possible approved methods to be adopted were evaluated from the samples using the phytoperiphyton, phytoplankton, zooplankton and zoobenthos against a background of hydrochemical characteristics.

Report on the analysis of salmon scale samples returned by anglers on the Rivers Ribble and Hodder

Fisheries technical staff from the North West Central Area of the National Rivers Authority (NRA) currently provide a service for the ageing of salmon scales from fish caught by anglers on the Rivers Ribble and Hodder in order to gather information on the biological characteristics of the salmon population on the River Ribble system in terms of weight, freshwater age and sea age. At the beginning of each fishing season, scale envelopes are distributed by the NRA to angling clubs and some individual anglers. Scales taken from salmon caught on the rivers are returned to the NRA Central Area Office by the anglers, or more often, by NRA bailiffs. The age of each fish caught is then determined by the identification and counting of annuli for both the river and sea zones on the scale. Information is provided by the angler on the scale packet concerning the length and weight of the fish caught, and the date, location and method of capture. Both this information and the age of the fish is recorded on a database. These data can be used to investigate the distribution, and exploitation patterns of the different age classes of the salmon stock within the river system. This report is principally concerned with the scale samples received in 1994, although comparison is also made with samples from 1993 and 1992. References to data will all relate to that received in 1994 unless an alternate year is stated.

Biological report - fish mortality, River Conder, 21.7.79

Following a large scale fish mortality caused by a considerable spillage of a pentachlbrophenol-based disinfectant from a mushroom farm, a biological survey of the river Conder was undertaken on 23.7.79. A spillage of this same make of disinfectant from this mushroom farm in February 1976 caused a similar large scale fish mortality. On that occasion, the pollution appeared to have little effect on the benthic invertebrates of the river Conder, with no dead organisms found, although the small beck which received the spillage was quite badly affected. This recent spillage of disinfectant involved a considerably greater volume (approximately 900 gallons) than the 1976 discharge and so this report looks at whether invertebrate fauna of the main river were affected on this occasion. Kick samples were taken at a number of sites in the river Conder from the confluence with the polluted beck to Conder Green. A control sample was also taken upstream of the confluence with the polluted beck. All samples were examined on site and live and dead invertebrates noted and identified as accurately as possible.

Species compositions of elasmobranchs caught by three different commercial fishing methods off southwestern Australia, and biological data for four abundant bycatch species

Commercial catches taken in southwestern Australian waters by trawl fisheries targeting prawns and scallops and from gillnet and longline fisheries targeting sharks were sampled at different times of the year between 2002 and 2008. This sampling yielded 33 elasmobranch species representing 17 families. Multivariate statistics elucidated the ways in which the species compositions of elasmobranchs differed among fishing methods and provided benchmark data for detecting changes in the elasmobranch fauna in the future. Virtually all elasmobranchs caught by trawling, which consisted predominantly of rays, were discarded as bycatch, as were approximately a quarter of the elasmobranchs caught by both gillnetting and longlining. The maximum lengths and the lengths at maturity of four abundant bycatch species, Heterodontus portusjacksoni, Aptychotrema vincentiana, Squatina australis, and Myliobatis australis, were greater for females than males. The L50 determined for the males of these species at maturity by using full clasper calcification as the criterion of maturity did not differ significantly from the corresponding L50 derived by using gonadal data as the criterion for maturity. The proportions of the individuals of these species with lengths less than those at which 50% reach maturity were far greater in trawl samples than in gillnet and longline samples. This result was due to differences in gear selectivity and to trawling being undertaken in shallow inshore waters that act as nursery areas for these species. Sound quantitative data on the species compositions of elasmobranchs caught by commercial fisheries and the biological characteristics of the main elasmobranch bycatch species are crucial for developing strategies for conserving these important species and thus the marine ecosystems of which they are part.

Biochemical biomarkers in samples processed by different methods in Environmental Specimen Banks

In the last decades the creation of new Environmental Specimen Banks (ESB) is increasing due to the necessity of knowing the effects of pollutants in both the environment and human populations. ESBs analyze and store samples in order to understand the effects of chemicals, emerging substances and the environmental changes in biota. For a correct analysis of the effect induced by these variables, there is a need to add biological endpoints, such as biomarkers, to the endpoints based on chemical approaches which have being used until now. It is essential to adapt ESB´s sampling strategies in order to enable scientists to apply new biological methods. The present study was performed to obtain biochemical endpoints from samples stored in the BBEBB (Biscay Bay Environmental Biospecimen Bank) of the Marine Station of Plentzia (PIE - UPV/EHU). The main objective of the present work was to study the variability caused in biochemical biomarkers by different processing methods in mussels (Mytilus galloprovincialis) from two localities (Plentzia and Arriluze) with different pollution history. It can be concluded that the selected biomarkers (glutathione S-transferase and acetylcholinesterase) can be accurately measured in samples stored for years in the ESBs. The results also allowed the discrimination of both sampling sites. However, in a further step, the threshold levels and baseline values should be characterized for a correct interpretation of the results in relation to the assessment of the ecosystem health status.

Biological parameters, body length-weight and length-height relationships for various species in Greek waters

Biological/fisheries parameters (L sub(oo) M, F) are presented for four fish species (Gadiculus argenteus; Gaidropsarus mediterraneous; Symphurus ligulatus; Lepidorhombus boscii) as well as body length-weight and length-height relationships for 11 and 12 fish species, respectively, estimated from trawl samples collected using three different cod-ends (stretched mesh size: 14 mm and 20 mm diamond-shaped and 20 mm square-shaped) during 1993-1994, in the western Aegean and North Euboikos Gulf, Greece. The fisheries paramaters, estimated from length-frequency using the ELEFAN approach and software, are discussed in the light of recent information on the selectivity of the presently used trawl cod-end (14 mm diamond shaped)

Intensive biological survey of the Glaze Brook catchment May 1981

This is the Intensive biological survey of the Glaze Brook catchment May 1981 report produced by the North West Water Authority in 1981. The aim of this report is to identify those pollution problems not identified during the routine biological water quality surveys, and to check the suitability of the routine biological sampling point. This report looks at an intensive biological water quality survey of the Glaze Brook catchment which was carried out by Biol. (S) on 13th-15th May, 1981. Kick samples of invertebrates were taken at 5 sites and all invertebrates were identified and counted in the field. The most significant water quality observations in the report were from: Borsdane brook, Pennington brook, Glaze brook, Amberswood brook, Cunningham brook, Old Mill brook, Shakerley brook, Astley brook and Shaw brook.

Biological survey: River Teign and tributaries with references to the discharge of ball clay wastes

This is the Biological survey of River Teign and tributaries with references to the discharge of ball clay wastes, document produced by South West Water Authority in 1973. This report focuses on on-site assessment of the benthic macro-invertebrates of the lower River Teign, River Bovey, Ugbrooke Stream and Blatchford Brook was undertaken on 19th and 22nd August 1977 to assess the effects of discharges from ball clay mining premises. Inspection of aquatic fauna in the field is of necessity, less extensive than laboratory identification of sorted samples, so that the resulting data underestimates the water quality. This is more pronounced in the Chandler Score than in the Trent Biotic Index which does not take account of species abundance. Chandler should thus be interpreted by the trends described rather than by absolute values calculated.

Seasonal growth of King George whiting (Sillaginodes punctata) estimated from length-at-age samples of the legal-size harvest

Samples of 11,000 King George whiting (Sillaginodes punctata) from the South Australian commercial and recreational catch, supplemented by research samples, were aged from otoliths. Samples were analyzed from three coastal regions and by sex. Most sampling was undertaken at fish processing plants, from which only fish longer than the legal minimum length were obtained. A left-truncated normal distribution of lengths at monthly age was therefore employed as model likelihood. Mean length-at-monthly-age was described by a generalized von Bertalanffy formula with sinusoidal seasonality. Likelihood standard deviation was modeled to vary allometrically with mean length. A range of related formulas (with 6 to 8 parameters) for seasonal mean length at age were compared. In addition to likelihood ratio tests of relative fit, model selection criteria were a minimum occurrence of high uncertainties (>20% SE), of high correlations (>0.9, >0.95, and >0.99) and of parameter estimates at their biological limits, and we sought a model with a minimum number of parameters. A generalized von Bertalanffy formula with t0 fixed at 0 was chosen. The truncated likelihood alleviated the overestimation bias of mean length at age that would otherwise accrue from catch samples being restricted to legal sizes.