Microplate Technique Research Articles

Evaluation of Fluoromycobacteriophages for Detecting Drug Resistance in Mycobacterium tuberculosis

We tested a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). After promising results in preliminary studies, the EGFP-phage was used to detect isoniazid (INH), rifampin (RIF), and streptomycin (STR) resistance in 155 strains of M. tuberculosis, and the results were compared to the resazurin microplate technique, with the proportion method serving as the reference standard. The resazurin technique yielded sensitivities of 94% for INH and RIF and 98% for STR and specificities of 97% for INH, 95% for RIF, and 98% for STR. The sensitivity of EGFP-phage was 94% for all three antibiotics, with specificities of 90% for INH, 93% for RIF, and 95% for STR. The EGFP-phage results were available in 2 days for RIF and STR and in 3 days for INH, with an estimated cost of ∼2$ to test the three antibiotics. Using a more stringent criterion for resistance improved the specificity of the EGFP-phage for INH and RIF without affecting the sensitivity. In preliminary studies, the EGFP-phage could also effectively detect resistance to the fluoroquinolones. The EGFP-phage method has the potential to be a valuable rapid and economic screen for detecting drug-resistant tuberculosis if the procedure can be simplified, if it can be adapted to clinical material, and if its sensitivity can be improved.

Susceptibility of Escherichia coli isolated from uteri of postpartum dairy cows to antibiotic and environmental bacteriophages. Part I: Isolation and lytic activity estimation of bacteriophages

The objective of this study was to isolate bacteriophages from environmental samples of 2 large commercial dairy farms using Escherichia coli isolated from the uteri of postpartum Holstein dairy cows as hosts. A total of 11 bacteriophage preparations were isolated from manure systems of commercial dairy farms and characterized for in vitro antimicrobial activity. In addition, a total of 57 E. coli uterine isolates from 5 dairy cows were phylogenetically grouped by triplex PCR. Each E. coli bacterial host from the uterus was inoculated with their respective bacteriophage preparation at several different multiplicities of infections (MOI) to determine minimum inhibitory MOI. The effect of a single dose (MOI=102) of bacteriophage on the growth curve of all 57 E. coli isolates was assessed using a microplate technique. Furthermore, genetic diversity within and between the different bacteriophage preparations was assessed by bacteriophage purification followed by DNA extraction, restriction, and agarose gel electrophoresis. Phylogenetic grouping based on triplex PCR showed that all isolates of E. coli belonged to phylogroup B1. Bacterial growth was completely inhibited at considerably low MOI, and the effect of a single dose (MOI=102) of bacteriophage preparations on the growth curve of all 57 E. coli isolates showed that all bacteriophage preparations significantly decreased the growth rate of the isolates. Bacteriophage preparation 1230–10 had the greatest antimicrobial activity and completely inhibited the growth of 71.7% (n=57) of the isolates. The combined action of bacteriophage preparations 1230–10, 6375–10, 2540–4, and 6547–2, each at MOI=102, had the broadest spectrum of action and completely inhibited the growth (final optical density at 600nm ≤0.1) of 80% of the E. coli isolates and considerably inhibited the growth (final optical density at 600nm ≤0.2) of 90% of the E. coli isolates. Restriction profile analysis demonstrated that all 4 phage preparations contained bacteriophages that were genetically distinct from each other according to the banding pattern of the fragments. The combination of several different bacteriophages can improve the spectrum of action, and the results of this study suggest that bacteriophages 1230–10, 6375–10, 2540–4, and 6547–2 should be used in combination as a cocktail.

Antibacterial and antioxidant activities of four kaempferol methyl ethers isolated from Dodonaea viscosa Jacq. var . angustifolia leaf extracts

Fractionation of dichloromethane and acetone fractions obtained by serial extraction from the leaf powder of Dodonaea viscosa Jacq. var. angustifolia (Sapindaceae) resulted in the isolation of four kaempferol methyl ethers. The compounds were identified by spectral data ( 1H NMR, 13C NMR and MS) as: 3, 5, 7-trihydroxy-4'-methoxyflavone ( 1); 5, 7, 4'-trihydroxy-3, 6-dimethoxyflavone ( 2); 5, 7-dihydroxy-3, 6, 4'-trimethoxyflavone (santin) ( 3); and 5-hydroxy -3, 7, 4'-trimethoxyflavone ( 4) together with 3,4',5,7-tetrahydroxy flavone (kaempferol) ( 5). Antioxidant potential of the compounds was evaluated using a DPPH spectrophotometric assay, while antibacterial activity was determined using a serial dilution microplate technique. The isolates demonstrated varying degrees of antioxidant and antibacterial activities. Of all the compounds investigated, compounds 1 and 5 demonstrated some antioxidant activity (EC 50 = 75.49 ± 1.76 µM and 35.06 ± 0.85 respectively) but lower than l-ascorbic acid (EC 50 = 13.55 ± 0.28 µM) used as a standard antioxidant agent. The minimum inhibitory concentration (MIC) of isolated compounds against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa varied from 16 µg/ml to more than 250 µg/ml. Some structure activity relationships could be established for these compounds.

Blood group serology

Blood group serology

Guidelines for microplate techniques in liquid-phase blood grouping and antibody screening

Summary. This document is an introduction to microplate serology prepared at a time of very active research and development. Antibody detection techniques are expected to improve during the lifetime of this document. Approximately 25% of hospital blood banks are ABO and D grouping by microplate techniques. Antibody screening by microplate techniques is only being carried out by a small percentage of hospital blood banks but there is likely to be a change to antiglobulin tests by microplate procedures as soon as the methods have been thoroughly tested and approved. There are one or two weak links in these procedures which could give rise to serious errors:— (1) Antiglobulin reagents standardized for spin-tube tests may be subject to prozones in liquid phase microplate tests. Each new batch of antiglobulin reagent must be standardized by the microplate procedure in use to show that the reagent is at its optimum anti-IgG dilution for use. However dilutions greater than 1:2 may seriously compromise the anti-complement activity of the reagent. (2) Automated cell washers for microplates are currently under development and should be evaluated by replicate tests with weak anti-D sensitized cells. (3) The combination of diluted anti-IgG and poor washing procedures may lead to neutralization of anti-IgG and cause false negative errors. Future development for improved antiglobulin tests in microplates by a solid phase system is now well advanced and workers proposing to change to antibody screening by microplates are advised to bear this in mind. It is hoped that microplate methodology will move towards standardization based on national guidelines using standard antibody reagents for the validation of new microplate procedures.

High‐throughput microplate technique for enzymatic hydrolysis of lignocellulosic biomass

Several factors will influence the viability of a biochemical platform for manufacturing lignocellulosic based fuels and chemicals, for example, genetically engineering energy crops, reducing pre-treatment severity, and minimizing enzyme loading. Past research on biomass conversion has focused largely on acid based pre-treatment technologies that fractionate lignin and hemicellulose from cellulose. However, for alkaline based (e.g., AFEX) and other lower severity pre-treatments it becomes critical to co-hydrolyze cellulose and hemicellulose using an optimized enzyme cocktail. Lignocellulosics are appropriate substrates to assess hydrolytic activity of enzyme mixtures compared to conventional unrealistic substrates (e.g., filter paper, chromogenic, and fluorigenic compounds) for studying synergistic hydrolysis. However, there are few, if any, high-throughput lignocellulosic digestibility analytical platforms for optimizing biomass conversion. The 96-well Biomass Conversion Research Lab (BCRL) microplate method is a high-throughput assay to study digestibility of lignocellulosic biomass as a function of biomass composition, pre-treatment severity, and enzyme composition. The most suitable method for delivering milled biomass to the microplate was through multi-pipetting slurry suspensions. A rapid bio-enzymatic, spectrophotometric assay was used to determine fermentable sugars. The entire procedure was automated using a robotic pipetting workstation. Several parameters that affect hydrolysis in the microplate were studied and optimized (i.e., particle size reduction, slurry solids concentration, glucan loading, mass transfer issues, and time period for hydrolysis). The microplate method was optimized for crystalline cellulose (Avicel) and ammonia fiber expansion (AFEX) pre-treated corn stover.

Determination of N-species in soil extracts using microplate techniques

Colourimetric methods for the determination of NO 3 −, NH 4 + and total N in water extracts of soils using 96-well microplate techniques are described. Nitrate was determined by azo dye formation after reduction to NO 2 − using a solution of hydrazine sulphate. Ammonium in the soil extracts was purified and concentrated by diffusion as NH 3 from small volumes (750 μL) of extract treated with MgO into a H 2SO 4 collector using a double-plate, MicroResp™ method and subsequently determined by the Berthelot reaction. For the determination of total N, samples were oxidised with K 2S 2O 8 at 110 °C in a 96 × 1.1 mL polytetrafluoroethylene block with a lid that closed individual wells. The oxidised solutions were transferred to standard plates for colourimetric analysis of NO 3 −. The recovery of N, measured as NO 3 −, from NH 4NO 3 and a range of organic-N compounds was >95%. The limits of quantitation of the colourimetic assays were 0.020 mg N L −1 for NO 3 − and 0.051 mg N L −1 for NH 4 +. The methods were tested on water extracts derived from a range of 10 nutrient poor soils from Scotland. There were acceptable linear correlations between the results obtained by established methods. For soil extracts analysed by the microplate method, the relationship for NO 3 − was 1.03× result from ion chromatography + 0.0055 ( R 2 = 0.9961); for NH 4 + determined by the microplate method, the relationship was 0.9696 × result from a discrete analyser − 0.0169 ( R 2 = 0.9757) and for total N determined by oxidation in the PTFE microplate the relationship was 0.9435 × result obtained by combustion + 0.0489 ( R 2 = 0.9743). Purification of the NH 4 + in water extracts from the 10 different soils by the diffusion method did not result in any systematic difference (paired t-test, p = 0.05) between measured concentration values determined before and after diffusion.

Effects of vegetation type on soil microbial community structure and catabolic diversity assessed by polyphasic methods in North China

Abstract Soil microbes play a major role in ecological processes and are closely associated with the aboveground plant community. In order to understand the effects of vegetation type on the characteristics of soil microbial communities, the soil microbial communities were assessed by plate counts, phospholipid fatty acid (PLFA) and Biolog microplate techniques in five plant communities, i.e., soybean field (SF), artificial turf (AT), artificial shrub (AS), natural shrub (NS), and maize field (MF) in Jinan, Shandong Province, North China. The results showed that plant diversity had little discernible effect on microbial biomass but a positive impact on the evenness of utilized substrates in Biolog microplate. Legumes could significantly enhance the number of cultural microorganisms, microbial biomass, and community catabolic diversity. Except for SF dominated by legumes, the biomass of fungi and the catabolic diversity of microbial community were higher in less disturbed soil beneath NS than in frequently disturbed soils beneath the other vegetation types. These results confirmed that high number of plant species, legumes, and natural vegetation types tend to support soil microbial communities with higher function. The present study also found a significant correlation between the number of cultured bacteria and catabolic diversity of the bacterial community. Different research methods led to varied results in this study. The combination of several approaches is recommended for accurately describing the characteristics of microbial communities in many respects.

A FLUOROMETRIC TECHNIQUE FOR THE IN VITRO MEASUREMENT OF GROWTH AND VIABILITY IN QUAHOG PARASITE UNKNOWN (QPX)

Quahog parasite unknown (QPX) is a protistan parasite that causes significant mortalities among hard clams, Mercenaria mercenaria, in the northeastern United States and Canada. This pathogen has been successfully isolated from clams from different geographic locations, and in vitro cultures are being used in investigations of the parasite's genetic makeup, virulence and environmental tolerances. Many of these investigations require an easily reproducible, quantitative method to rapidly measure QPX cell viability and proliferation. Therefore, a fluorometric microplate technique was developed using fluorescein diacetate (FDA) as an indicator of cell viability. The developed technique provides a good estimate of the biovolume of live QPX cells. Fluorescent signals are correlated with the number and the size of individual QPX cells. Optimal experimental conditions included an FDA concentration range of 30–50 μM with an incubation period of 15–30 min in the dark. This FDA assay was used to investigate the dynamics of in vitro growth of QPX. Results showed that the growth dynamic is different among QPX isolates. For instance, exponential growth lasted for 1 week in two QPX isolates cultured from clams collected from New York and Massachusetts; whereas a third isolate (from New York) grew exponentially during 2 weeks under similar experimental conditions. While there are limitations to in vitro studies that must be recognized, research using cultured QPX cells is indispensable and will probably lead to significant progress in our knowledge of the biology and physiology of this parasite.

Patterns of Sole-Carbon-Source Utilization by Fast-Growing Coastal Sand Dune Rhizobia of the Southwest Coast of India

Coastal sand dunes harbor a variety of free living and symbiotic microorganisms, which are adapted to stress conditions such as temperature, salinity and pH. The current paper explores the ability of sole-source-carbon utilization by symbiotic rhizobia isolated from tropical coastal sand dune wild legumes. Fast-growing rhizobia isolated from five legume plant species (Canavalia cathartica, Canavalia maritima, Crotalaria retusa, Crotalaria verrucosa, and Derris triflorum) grown on the two coastal sand dunes of the southwest coast of India (Someshwara, S1–S5; Padubidri, P1–P5) were assessed for sole-carbon-source utilization patterns based on BIOLOG GN2 microplate technique. All the isolates showed high extents of utilization of the different carbon sources within 24 h of incubation at 30 °C. Cluster analysis based on quantitative and qualitative utilization of a sole carbon source revealed two distinct clusters. Cluster I consists of four isolates (S1, P2, P4, and P5), of which P2 and P5 showed greater similarities. The cluster II encompasses six isolates (S2–S5, P1 and P3), among them S3 and S5 showed high similarities. Based on the utilization of six guilds by the isolates S1, S2, P1, and P2 (polymers, carbohydrates, carboxylic acids, amides and amines, amino acids and miscellaneous), S2 and P1 exhibited high functional diversity. Principal component analysis revealed a close catabolic potential between the isolates S5 and P1; S1 and P3; S3, P2, and P5. The symbiotic rhizobia of the southwest coastal sand dune wild legumes of India studied might serve as novel inoculants to achieve legume production through nitrogen fixation under the varied conditions of tropical soils. These rhizobia were able to utilize a wide range of carbon sources, possessed functional diversity, withstood extreme conditions (temperature, salinity, pH), exhibited non-host specificity and induced high biomass in edible legumes.

Suppression of Ca 2+ influx by unfractionated heparin in non-excitable intact cells via multiple mechanisms

Effect of unfractionated heparin (UFH), described as a cell-impermeant IP3 receptor antagonist, was studied on the capacitive Ca 2+ entry in non-permeabilized, intact cells, measuring the intracellular Ca 2+ levels using fluorescence microplate technique. Ca 2+ influx induced via Ca 2+ mobilization by histamine in Hela cells or evoked by store depletion with thapsigargin in RBL-2H3 cells was dose-dependently suppressed by UFH added either before or after the stimuli. UFH also prevented the spontaneous Ba 2+ entry indicating that the non-capacitive Ca 2+ channels may also be affected. In addition, UFH caused a significant and dose-dependent delay in Ca 2+, and other bivalent cation inflow after treatment of the cells with Triton X-100, but it did not diminish the amount of these cations indicating that UFH did not act simply as a cation chelator, but modulated the capacitive Ca 2+ entry possibly via store operated Ca 2+ channels (SOCCs). Inhibitory activities of UFH and 2-aminoethyl diphenyl borate on the capacitive Ca 2+ influx was found reversible, but the time courses of their actions were dissimilar suggesting distinct modes of action. It was also demonstrated using a fluorescence potentiometric dye that UFH had a considerable hyperpolarizing effect and could alter the changes of membrane potential during Ca 2+ influx after store depletion by thapsigargin. We presume that the hyperpolarizing property of this agent might contribute to the suppression of Ca 2+ influx. We concluded that UFH can negatively modulate SOCCs and also other non-capacitive Ca 2+ channels and these activities might also account for its multiple biological effects.

マイクロプレートを用いたＡＧＰ試験の検討

The algal growth potential (AGP) test is a useful tool for assessing the eutrophication of rivers and lakes. Nevertheless, the AGP test is relatively expensive because of its large scale. In this study, a microplate technique was used in the AGP test to develop a microscale version. The algal growth in the microplate wells was monitored by examining the relationship between the absorbance (450 nm) measured using a microplate reader and cell concentration. A high correlation was observed between absorbance and cell concentration. There were few differences between the microplate wells with respect to well position, suggesting that the effect of well position on algal growth is negligible. The microscale AGP test using a microplate was compared with the conventional flask-size AGP test. A high similarity between the two methods was observed. Therefore, the microscale AGP test is a simple and useful method for conducting AGP tests on many water samples. The change in AGP level in the Tama River was investigated. AGP level increased considerably after inflow of water discharged from sewage treatment plants (STPs). This study has demonstrated that STP discharge has a considerable effect on river water quality.

Environmentally low-temperature kinetic and thermodynamic study of lactate dehydrogenase from Atlantic cod ( G. morhua) using a 96-well microplate technique

Analyses of temperature-dependent kinetic parameters in enzymes extracted from tissues of ectothermic animals are usually carried out within the range of physiological temperatures (0–40 °C). However, multisample spectrophotometers (so-called microplate readers) with efficient wide-range temperature control (including cooling) have previously been unavailable. This limits the statistical quality of the measurements. A temperature-controlled microplate was designed for a 96-well microplate reader to overcome this limitation. This so-called T-microplate is able to control assay temperature between the freezing point of a liquid sample and 60 °C with high stability and accuracy in any data acquisition mode. At 4 °C the accuracy of the temperature control was ±0.1 °C and temperature homogeneity across the microplate was ±0.3 °C. As examples, analyses of the temperature dependence of Michaelis–Menten ( K ′PYR m) and substrate inhibition ( K si PYR) constants for pyruvate, of the maximal rate of reaction ( V ′ max), of the apparent Arrhenius activation energy ( E A), and of the Gibbs free-energy change ( ΔG ‡ ) of lactate dehydrogenases from muscle of Atlantic cod Gadus morhua acclimated to 4 °C are described. The large dataset obtained allowed evaluation of a new mechanism of metabolic compensation in response to seasonal temperature change.

Comparison of the efficacy of different techniques, culture media, and sources of blood in determining the hemolytic activity ofListeriaspp.

Hemolytic activity is a fundamental criterion for the differentiation of Listeria species; therefore, a simple and inexpensive procedure to clearly distinguish hemolytic strains from each other and from nonhemolytic strains would be of great aid. We compared the efficacy of several techniques, culture media, and types of blood in demonstrating the hemolysis of Listeria spp. The hemolytic activities of Listeria monocytogenes and Listeria seeligeri were more easily detected with a red blood cell top-layer (RBCTL) technique and with a microplate technique than when the strains were streaked on blood agar (BA). Listeria ivanovii produced a marked hemolysis regardless of the technique employed. In general, the hemolytic activity of these three species was stronger on media containing brain heart infusion (BHI) agar and (or) potassium tellurite (PT). However, Listeria innocua produced questionable hemolytic reactions when nonselective culture media with BHI and PT were utilized, limiting the advantages gained by employing the two compounds. The RBCTL and the BA techniques disclosed greater hemolytic activity for L. monocytogenes, L. seeligeri, and L. ivanovii with sheep and guinea pig blood than with horse and human blood. When the microplate technique was used, all four kinds of blood were equally effective.

Distribution and location of polycyclic aromatic hydrocarbons (PAHs) and PAH-degrading bacteria within polluted soil aggregates.

A study was conducted to determine the location and distribution of PAH and PAH-degrading bacteria in different aggregate size fractions of an industrially polluted soil. The estimation of PAH-degrading bacteria using an MPN microplate technique indicated that these bacteria are most numerous in the aggregate size fractions corresponding to fine silt (2-20 microm) and clay (<2 microm) compared to larger fractions or unfractionated soil. PAH concentrations were also highest in the aggregate size fraction corresponding to fine silt. Similar results were found in a spiked soil (incubated for 6 months) with similar carbonated minerals. Transmission electron microscopy observations showed that the autochtonous PAH-degrading bacteria were embedded in the aggregates where PAHs were abundant. In spite of this extensive co-localisation PAH degradation was limited during 6 months incubation. This indicates that factors other than spatial distribution and PAH degrading ability control degradation rates. The fine silt fraction of the industrial soil had an elevated C/N ratio (35) compared to the clay fraction (C/N: 16). Thus the fraction which assumably had the highest specific surface area contained less PAH but similar numbers of PAH-degraders. N thus seem to play an important role in the long term, but as PAH degradation was low in fine size fractions, other sources/factors were probably limiting (easily degradable C, P org, O2 etc.). Based on these findings, soil particle organization and structure of soil aggregates appear to be important for the characterization of a polluted soil (localization and sequestration). Manipulations that modify aggregation in polluted soils could thus potentially influence the accessibility and biodegradability of PAHs.

Effects of sulfonylurea herbicides on non-target aquatic micro-organisms: Growth inhibition of micro-algae and short-term inhibition of adenine and thymidine incorporation in periphyton communities

To study the differential sensitivity to sulfonylurea herbicides of aquatic micro-organisms we used the micro-plate technique as previously described by Blanck and Björnsäter [Blanck, H., Björnsäter, B., 1989. The algal microtest battery—a manual for routine tests of growth inhibition. Report to the Swedish National Chemicals Inspectorate, no 3/89]. This technique enables growth-inhibition tests to be carried out for a large number of species of micro-algae, and growth inhibition data for the two sulfonylureas chlorsulfuron and metsulfuron methyl were obtained for 20 marine species and 20 fresh-water species, respectively. The species-dependent variation in algal sensitivity was found to be very large, with strains of cyanobacteria and dinoflagellates being the most sensitive. The most sensitive species had EC 50 values for the inhibition of growth in the nanomolar range while EC 50 was higher than 1000 μM for the most tolerant species. Field experiments were also carried out to examine the sensitivity of periphyton communities in an agricultural area in southern Sweden. Periphyton communities were established on an artificial substratum in a creek affected by the runoff of agricultural pesticides and a creek with a watershed with no use of pesticides. Short-term toxicity of the sulfonylurea tribenuron methyl was assayed as inhibition of adenine and thymine incorporation weekly for 8 weeks at all sites. At the site with high exposure to pesticides and fertilisers the lowest observed effect concentration (LOEC) values for inhibition of adenine incorporation were always higher than environmentally expected concentrations indicating a high tolerance of periphyton to tribenuron methyl at this site. The toxicity of tribenuron methyl at the reference site was very variable over time. Adenine incorporation in periphyton from the reference site was in three out of seven experiments significantly inhibited at 3 nM tribenuron methyl, but in the remaining four experiments, LOEC values were much higher. The apparent high background variability in the short-term toxicity of tribenuron methyl makes an interpretation of the observed differences between the reference site and the site with higher exposure to pesticides very difficult. It is never the less interesting to compare the growth inhibition test data with some of the field toxicity tests at the reference site, showing that sulfonylurea herbicides can adversely affect both algal growth and periphyton activity at nanomolar concentrations.

Influence of microplate material on the sensitivity of growth inhibition tests with bacteria assessing toxic organic substances in water and waste water

In conjunction with aquatic bioassays, the effect of microplate (MP) material on the sensitivity of a cell multiplication inhibition test with luminescent bacteria (as representatives for unicellular organisms) was examined. The tested substances were four organic chemicals of different hydrophobic properties (4-nitrotoluene, 3,5-dichlorophenol, 2-chloroaniline, sodium phenolate) and waste water from a chemical production plant. The study revealed, that (except for sodium phenolate) the toxic response to the testing material was influenced by the MP material. The employment of the common plastic MP led to an underestimation of the biotests results. Therefore it must be taken into consideration, that when using the microplate technique to expose unicellular organisms to toxic chemicals with hydrophobic properties, only MP made of quartz glass should be employed. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 424–428, 1999

Development of a Magnetic Microplate Chemifluorimmunoassay for Rapid Detection of Bacteria and Toxin in Blood

A magnetic microplate chemifluorimmunoassay (MMCIA) is described using an immunomagnetic separation and a fluorescent microplate technique for rapid detection of low-levelEscherichia coliO157:H7,Bacillus subtilisvar.nigerspores, andStaphylococcal enterotoxintype B from whole blood. In general, the MMCIA has at least several-fold more sensitivity than the conventional enzyme-linked immunosorbent assay. In addition, the assay sensitivities using direct fluorochrome label as the reporter, or alkaline phosphatase (AP) with various assay substrates, such as pNPP and AttoPhos, were assessed.

Assessment of Equipment for the Determination of Nutrients in Marine Waters: A Case Study of the Microplate Technique

An essential prerequisite for quality assurance of the colorimetric determination of nutrients in seawater is the use of suitable photometric equipment. Based on a knowledge of the optical characteristics of a particular system and the absorption coefficient of the analyte, a statistical approach can be used to predict the limit of detection and the limit of quantitation for a given determinand. The microplate technique, widely used for bioassays, is applicable to colorimetric analysis in general, and its use for the determination of nutrients in seawater has been suggested. This paper reports a theoretical assessment of its capabilities in this context and a practical check on its performance, taking the determination of nitrite in seawater as typical. The conclusion is that short optical path length and insufficient repeatability of the absorbance measurement render it unsuitable for the determination of the low concentrations generally encountered in marine work, with the possible exception of nitrate. The perceived advantage of high-speed analysis is a secondary consideration in the overall process of determining nutrients, and the microplate technique's small scale of operation is a definite disadvantage as this increases the risk of exposure to contamination problems, in comparison with conventional techniques.

Development and application of a simple procedure for toxicity testing using immobilized algae

A simple microplate technique was adopted for toxicity assessment of a number of pesticides including six herbicides (Atrazine, Dichloroprop, Glyphosphate, Chlorsulfuron, MCPA, and Simazine), an insecticide (Dimethoate) and a fungicide (Propiconazol). Growth response of free and immobilized cultures of the green chlorococcal algae Selenastrum capricornumtum to different treatments of these pesticides was tested and compared. The biotests were carried out under conditions optimal for the growth of the test alga. Algal growth was exposed in terms of dry weight, and was employed as the toxicity-response parameter. Dose-response curves were used to calculate the toxicity of the tested compounds in terms of EC50. Based on EC50 values, the responses of both immobilized and free cultures were quite similar for almost all the treatments. The technique facilitated the visual detection of the lowest toxic concentration giving no detectable algal growth (EC100). The technique is quite simple, rapid, practical, accurate, and space saving. It suggested that batteries of immobilized algae could replace free cultures in studies of toxicity testing.