Analysis of nitrate in food extracts using a thermostable formate linked nitrate reductase enzyme system

Summary A method for reduction of nitrate to nitrite for determination of nitrate is described, using a thermostable formate linked nitrate reductase enzyme system (FLNR). The reduction of nitrate to nitrite was found to be quantitative in water and in various food samples containing nitrate. The method is suggested as an alternative for the cadmium reduction method.     

An automated fluorescence assay for subnanogram quantities of protein in the presence of interfering material

An automated assay for measuring nanogram and subnanogram quantities of protein in microliter samples was developed with the fluorometric reagent omicron-phthaldialdehyde/mercaptoethanol. Low molecular weight interfering substances were separated within the analysis by gel filtration. The technique allowed measurement of biological fluids without any sample pretreatment. The method presented proved to be linear within the range 1.2 ng to 1.4 microgram with a standard deviation of +/- 4.2%. The minimum detectable protein concentration was 1 microgram/liter. Special care was taken to prevent any determination errors caused by losses of protein during adsorption to surfaces. The simple analytical apparatus constructed can be used for field studies and ran several weeks when continuously used for seawater analysis aboard ship.     

Mechanism of Prophylaxis by Silver Compounds against Infection of Burns

To clarify tthe mechanism by which local application of silver compounds protects burns against infection, an ion-specific electrode was used to measùre the concentration of silver ions in solutions. By this method it was shown that in burn dressings silver ions were reduced to a very low level by precipitation as silver chloride. The antibacterial effect was found to depend on the availability of silver ions from solution in contact with precipitate. Between 10^-5 and 10^-6 molar silver nitrate solution in water was rapidly bactericidal. The minimal amount of silver nitrate causing inhibition of respiration of skin in tissue culture was about 25 times the minimal concentration of silver nitrate that inhibited growth of Pseudomonas aeruginosa.     

含无机盐沉淀菌悬液中微生物生长量的快速测定

目的:消除菌悬液中无机盐沉淀对比浊法测定生长量的干扰。方法:以嗜有机甲基杆菌ME25为材料,采用比浊法,研究了EDTA对菌悬液中无机沉淀物的清除效应以及对菌体生物量测定的影响。结果:在室温、pH 4~11,1.25×10^-2 mol/L EDTA与菌悬液样品作用1min,即可去除样品中无机盐沉淀,样品稳定,在1h内不影响样品中菌体吸光度的测定;实际样品和理论样品测定,相对误差小于3.0%,回收率为98%~100%,RSD均小于0.5%。结论:采用螯合剂EDTA可快速去除菌悬液中的无机盐沉淀,有效地消除沉淀物的干扰,明显提高了比浊法测定生长量的准确度,简便易行,具有较高的实用价值。     

An optical method for the rapid measurement of micromolar concentrations of nitrate in marine phytoplankton cultures

This method of nitrate determination by ultraviolet absorption spectrometry is based on the measurement of sample absorbance at a single wavelength (220 nm), which was chosen on the basis of the absorption spectra of the main components of artificial seawater in the ultraviolet domain. No reagents are used and no sophisticated instruments are necessary. For standards prepared in artificial seawater, the relationship between absorbance and nitrate concentration is linear up to 500 μmol N L⁻¹ and the detection limit is 1 μmol N L⁻¹. Precision is 1.5%. Urea and amino acids did not interfere at concentrations typical of seawater. The method also measures nitrite, but this interference only becomes important for species which excrete large amounts of nitrite. The method is extremely rapid, simple to implement and does not require the use of toxic chemicals such as cadmium. It should prove useful for monitoring quickly the nitrate concentrations in laboratory cultures of marine phytoplankton. This revised version was published online in July 2006 with corrections to the Cover Date.     

The tolerance of grass carp Ctenopharyngodon idella (Val.) to seawatert†

Laboratory tests were conducted to determine the tolerance of 2+ grass carp to an oceanic water. It was found that at a temperature of 20.1°C±0.4°C the maximum survival times of the fish in concentrations of seawater equivalent to 17.5 and 10.5 g/1 sodium chloride were 5 h and approximately 24 days respectively. The importance of these results lies in the consideration of the possibility of grass carp migrating through brackish or seawater from one river system to another. Such a migration would seem to be impossible through seawater but may be possible through brackish water.     

Cortisol receptor blockade and seawater adaptation in the euryhaline teleost Fundulus heteroclitus

To examine the role of cortisol in seawater osmoregulation in a euryhaline teleost, adult killifish were acclimated to brackish water (10 per thousand) and RU486 or vehicle was administered orally in peanut oil daily for five days at low (40 mg.kg(-1)) or high dose (200 mg.kg(-1)). Fish were transferred to 1.5 x seawater (45 per thousand) or to brackish water (control) and sampled at 24 h and 48 h after transfer, when Cl- secretion is upregulated. At 24 h, opercular membrane Cl- secretion rate, as Isc, was increased only in the high dose RU486 group. Stimulation of membranes by 3-isobutyl-1-methylxanthine and cAMP increased Isc in vehicle treated controls but those from RU486-treated animals were unchanged and membranes from brackish water animals showed a decrease in Isc. At 48 h, Isc increased and transepithelial resistance decreased in vehicle and RU486 groups, compared to brackish water controls. Plasma cortisol increased in all groups transferred to high salinity, compared to brackish water controls. RU486 treated animals had higher cortisol levels compared to vehicle controls. Vehicle treated controls had lower cortisol levels than untreated or RU486 treated animals, higher stimulation of Isc, and lower hematocrit at 24 h, beneficial effects attributed to increased caloric intake from the peanut oil vehicle. Chloride cell density was significantly increased in the high dose RU486 group at 48 hours, yet Isc was unchanged, suggesting a decrease in Cl- secretion per cell. Thus cortisol enhances NaCl secretion capacity in chloride cells, likely via glucocorticoid type receptors.     

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

The osmorespiratory compromise in sculpins: impaired gas exchange is associated with freshwater tolerance

We acclimated two species of sculpin, the freshwater prickly sculpin (Cottus asper) and the closely related marine Pacific staghorn sculpin (Leptocottus armatus) to freshwater ( approximately 0 g/L), brackish water (15 g/L), and seawater (30 g/L) for at least 4 wk and examined the relationships between respiration, ion regulation, gill morphology, and freshwater tolerance. The prickly sculpin successfully acclimated to all three salinities and did not experience appreciable changes in plasma osmolality, [Cl-], or mortality. Gill Na+/K+-ATPase activity was lowest in prickly sculpins acclimated to freshwater, their native salinity, and increased during acclimation to seawater. Furthermore, prickly sculpins acclimated to freshwater had a 30% higher P(crit) than fish acclimated to brackish water or seawater; P(crit) is the environmental P(O2) below which an animal can no longer maintain a routine (.-)M(O2), and an increase in P(crit) represents a compromise of respiratory gas exchange. The higher P(crit) observed in prickly sculpins acclimated to freshwater is likely a consequence of their having small, relatively thick gills that increase in thickness (by approximately 1 microm) during freshwater exposure. In contrast, the marine Pacific staghorn sculpin successfully acclimated to brackish water and seawater, but high mortality (25%) was observed after 3 wk of exposure to freshwater. Pacific staghorn sculpins exposed to freshwater suffered significant, 15%-20%, reductions in plasma osmolality and [Cl-], and these losses in plasma ions resulted in a 1.4-fold increase in gill Na+/K+-ATPase activity. Pacific staghorn sculpins have large, thin gills that are not modified in response to salinity acclimation, and as a result, these animals show no respiratory compromise during freshwater acclimation, as evidenced by the lack of change in P(crit), but show significant ion regulatory disturbance. Overall, this study suggests that gill thickening and the resulting respiratory compromise are necessary for freshwater tolerance in sculpins.     

Multiple cosmopolitan ecotypes within a microbial eukaryote morphospecies

Microbial eukaryotes that are morphologically indistinguishable (i.e. 'morphospecies') tend to be genetically diverse. While most protist morphospecies have cosmopolitan distribution, it has been suggested that ribotypes (unique rRNA gene sequences) or rRNA sequence clusters do have biogeography and such clusters may correlate with particular (non-morphological) adaptations. We have studied this in the ciliated protozoan morphospecies Cyclidium glaucoma. Fifty-four isolates collected worldwide represented 31 distinct ribotypes. There was no evidence of biogeographic distribution patterns. For example, identical ribotypes occurred in samples from Argentina, Peru, Morocco, Russia and Ukraine; in samples from Denmark and Australia; and in samples from Great Salt Lake and hyperhaline ponds in Spain. The morphospecies Cyclidium glaucoma is euryhaline and occurs in freshwater, brackish water, seawater, and hyperhaline waters. Evidence suggests that one ribotype cluster occurs only in marine or brackish habitats, and another one has so far been found only in hyperhaline habitats. Two clades seem to occur only in freshwater, but one clade includes ribotypes that were found in freshwater as well as in brackish water.     

Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy

A macro and micro assay for the spectrophotometric determination of serum nitrite and nitrate was developed. Nitrite/nitrate in biological samples can be estimated in a single step by this method. The principle of the assay is the reduction of nitrate by copper-cadmium alloy, followed by color development with Griess reagent (sulfanilamide and N-naphthylethylenediamine) in acidic medium. This assay is sensitive to 1 microM nitrate and is suitable for different biological fluids, including sera with a high lipid concentration. The copper-cadmium alloy used in the present method is easy to prepare and can completely reduce nitrate to nitrite in an hour. The present method provides a simple, cost-effective assay for the estimation of stable oxidation products of nitric oxide in biological samples.     

Determination of complexation capacity using coulometric stripping analysis

Abstract

Complexation capacity is a measure of the ability of a water sample to complex metal ions. In this work, a new method which involves coulometrically plating copper on to a glassy carbon electrode and then completely stripping it into a vigorously stirred sample solution has been used to determine the complexation capacity. We have called this technique coulometric stripping analysis (CSA). A technique which uses several electrodes with different amounts of plated copper can be used to determine the complexation capacity. The accuracy of the method was examined using EDTA standard solutions and the method was applied to analyse some water samples.     

Paleoceanography of the Late Cretaceous (Maastrichtian) Western Interior Seaway of North America: evidence from Sr and O isotopes

Well-preserved fossils of the Late Cretaceous Western Interior Seaway (WIS) of North America have been analyzed for Sr concentration and Sr and O isotopes in order to decipher paleosalinities and paleotemperatures. The samples are from four biofacies within the Seaway (late Maastrichtian): offshore Interior (Pierre Shale), nearshore Interior (Fox Hills Formation), brackish (reduced salinity; Fox Hills Formation) and freshwater (Hell Creek Formation). Samples were also obtained from the Severn Formation of Maryland (considered to be representative of the open ocean). All biofacies (except the freshwater) are demonstrably within the Jeletzkytes nebrascensis ammonite zone (<1 Ma duration). The ⁸⁷Sr/⁸⁶Sr ratios show significant and systematic decreases from marine (mean±1 S.D.=0.707839±0.000024) to brackish facies (mean±1 S.D.=0.707677±0.000036), consistent with dilution by freshwater with a lower ⁸⁷Sr/⁸⁶Sr ratio than seawater. Such variation disallows using the ⁸⁷Sr/⁸⁶Sr ratios of fossil shell material to assign ages to fossils from the Late Cretaceous WIS without knowledge of the salinity in which the organism grew. The Sr isotope ratios for scaphitid ammonites within a single biofacies are similar to each other and different from those for scaphites in other biofacies, implying that these organisms are restricted in their distribution during life. The ⁸⁷Sr/⁸⁶Sr values of freshwater unionid mussels range widely and are not compatible with the freshwater endmember ⁸⁷Sr/⁸⁶Sr ratio required by the trend in ⁸⁷Sr/⁸⁶Sr vs. biofacies established from the other samples. Paleosalinities for the biofacies are estimated to range from 35‰ in the open marine to a minimum of 20‰ in the brackish, based on the presence of cephalopods in all four facies and the known salinity tolerance of modern cephalopods. Producing reasonable ⁸⁷Sr/⁸⁶Sr values for the freshwater endmember of a ⁸⁷Sr/⁸⁶Sr vs. 1/[Sr] plot requires a Sr concentration 0.2-0.5 that of seawater for the dominant freshwater input to the WIS. Such high Sr concentrations (relative to seawater) are not observed in modern rivers, and we suggest that the brackish environment in the WIS arose through the mixing of freshwater and seawater in a nearshore aquifer system. Reactions of the solution with aquifer solids in this ‘subterranean estuary’ [Moore, Mar. Chem. 65 (1999) 111-125] produced brackish water with the Sr concentration and isotopic composition recorded in the brackish biofacies. δ¹⁸O values of the fossils show decreases from the marine to brackish biofacies consistent with increasing temperatures (from ∼13 to 23°C) or, if temperatures were relatively constant, to a decrease in the δ¹⁸O of the water in which the shell formed. The latter interpretation is consistent with less-than-fully marine salinities in the nearshore biofacies, but both changes in temperature and the isotopic composition of the water may have occurred in this environment.     

A comparison of 35S-SO(4)(2-) radiotracer techniques to determine sulphate reduction rates in laminated sediments

In order to find a simple and efficient method to determine sulphate reduction rates in environmental samples, we tested different 35S-SO(4)(2-) radiotracer techniques. The methods varied in the application of 35S-SO(4)(2-) and subsequent extraction of reduced 35S-sulphur species. Samples were either incubated as sediment slurries mixed with the radiotracer, or as undisturbed sediment cores after core injection of the radiotracer. Reduced 35S-sulphur species were retrieved passively by diffusion or actively by reflux distillation. The methods were applied to surface sediments derived from three aquatic habitats situated in Germany: (1) a tideless brackish water, (2) a mining lake and (3) a natural freshwater lake. The best possible method was expected to yield the highest sulphate reduction rates, which were reproducible with respect to magnitude and depth distribution. At the same time, we aimed to keep the disturbance of samples as well as the expenditure of labour and equipment to a minimum. For all three types of aquatic habitats, the combination of core injection followed by diffusion was the most reliable and efficient method. This combination is therefore recommended for determination of sulphate reduction rates in laminated sediments.     

A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

盐分对河口淡水、微咸水沼泽湿地土壤甲烷产生潜力的影响

海平面上升导致河口区盐水入侵现象日益明显,深刻影响着河口潮汐淡水、微咸水湿地生物地球化学循环。采集闽江河口区淡水、微咸水短叶茳芏潮汐沼泽湿地表层土样,室内添加盐度为5,10,15,21 g/L的人造海水、Na Cl溶液及盐度为0的去离子水,通过室内泥浆厌氧培养试验,对比研究海水和Na Cl溶液对淡水、微咸水沼泽湿地土壤甲烷产生潜力的影响。与对照相比,1—12 d培养期内4个盐度的海水处理均显著抑制河口淡水、微咸水沼泽湿地甲烷产生潜力,抑制率在93%以上,盐度10—21 g/L的3个海水处理对于河口淡水、微咸水沼泽湿地甲烷产生潜力的抑制效应无显著差异。Na Cl溶液只有在盐度达到15和21 g/L才显著抑制淡水、微咸水沼泽湿地甲烷产生潜力,且抑制率最多为80.9%,盐度为5、10 g/L的Na Cl溶液对淡水、微咸水沼泽湿地甲烷产生潜力的抑制作用不显著,抑制率多小于30%。伴随着盐水入侵而发生的硫酸盐还原作用及离子胁迫作用对河口淡水、微咸水沼泽湿地甲烷产生具有显著的抑制效应。     

Colorimetric determination of chloride in biological samples by using mercuric nitrate and diphenylcarbazone

A colorimetic method is outlined for the determination of the chloride ion in biological samples (blood serum, plasma, and urine). The present method is based on the quantitative reduction of free mercuric ions by chloride ions. Chloride ions form an indissociable complex with mercuric ions. The remaining free mercuric ions form a purple complex with diphenylcarbazone with an absorption maximum at 550 nm. The reduction of color intensity at 550 nm is directly proportional to chloride concentration in the sample. The linear concentration range in the final reaction mixture was 0–100 μM with a correlation coefficient of −0.9997. The coefficient of variation for the 50 μM chloride ion in the final reaction mixture was 0.9% (n=6). The analyzed value of chloride concentration in the human control serum Accutrol™ Normal (Sigma) was 101±4 mM (mean±SD, n=12). The certified value of chloride in Accutrol Normal by Sigma is 102 mM, with a mean in the range 91–113 mM. This method was applied to the measurement of urinary chloride excretion in experimental rats. During 16-h urine collection, no food was given and rats had free access to purified water. The urinary excretion rate of chloride was 23.6±9.3 μmol/h (mean±SD, n=8) and 126.2±28.0 μmol/h (n=8) for rats fed a normal diet (2.6 g NaCl/kg diet) and a high-salt diet (82.6 g NaCl/kg diet) for 70 d prior to urine collection, respectively. This method is appropriate for low concentrations of chloride in samples or when sample volume is limiting, as in many animal studies such as metabolic urine collection from rats. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of the products that may also be suitable.     

Microbial response to reinjection of produced water in an oil reservoir

The microbial response to produced water reinjection (PWRI) in a North Sea oil field was investigated by a combination of cultivation and culture-independent molecular phylogenetic techniques. Special emphasise was put on the relationship between sulphate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB), and results were used to evaluate the possibility of nitrate treatment as a souring management tool during PWRI. Samples were collected by reversing the flow of the injection water, which provided samples from around the injection area. The backflowed samples were compared to produced water from the same platform and to backflowed samples from a biocide-treated seawater injector, which was the previous injection water treatment of the PWRI well. Results showed that reinjection of produced water promoted growth of thermophilic SRB. Thermophilic fatty acid oxidising NRB and potential nitrate-reducing sulphide-oxidising bacteria were also found. The finding of thermophilic NRB makes nitrate treatment during PWRI possible, although higher nitrate concentration will be necessary to compensate for the increased SRB activity.     

Monitoring of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase mRNA expression in the cinnamon clownfish, <Emphasis Type="Italic">Amphiprion melanopus</Emphasis>, exposed to an osmotic stress environment: profiles on the effects of exogenous hormone

We examined changes in the expression of Na⁺/K⁺-ATPase mRNA in the gills of the cinnamon clownfish using quantitative real-time PCR in an osmotically changing environment [seawater (35 psu; practical salinity unit, 1 psu ≈ 1‰) → brackish water (17.5 psu) and brackish water with prolactin]. The expression of Na⁺/K⁺-ATPase mRNA in gills was increased after the transfer to brackish water, and the expression was repressed by prolactin treatment. Also, activities of gill Na⁺/K⁺-ATPase and plasma cortisol levels increased after the transfer to brackish water and were repressed in brackish water with prolactin treatment. Na⁺/K⁺-ATPase-immunoreactive cells were almost consistently observed in the gill filaments, but absent from the lamella epithelia. The plasma osmolality level decreased in brackish water, but the level of this parameter increased in brackish water with prolactin treatment during salinity change. These results suggest that the Na⁺/K⁺-ATPase gene plays an important role in osmoregulation in gills, and prolactin improves the hyperosmoregulatory ability of cinnamon clownfish in a brackish water (hypoosmotic) environment.     

Determination of the Viability of Aeromonas hydrophila in Different Types of Water by Flow Cytometry, and Comparison with Classical Methods

The presence of Aeromonas spp. in water can represent a risk for human health. Therefore, it is important to know the physiological status of these bacteria and their survival in the environment. We studied the behavior of a strain of Aeromonas hydrophila in river water, spring water, brackish water, mineral water, and chlorinated drinking water, which had different physical and chemical characteristics. The bacterial content was evaluated by spectrophotometric and plate count techniques. Flow cytometric determination of viability was carried out using a dual-staining technique that enabled us to distinguish viable bacteria from damaged and membrane-compromised bacteria. The traditional methods showed that the bacterial content was variable and dependent on the type of water. The results obtained from the plate count analysis correlated with the absorbance data. In contrast, the flow cytometric analysis results did not correlate with the results obtained by traditional methods; in fact, this technique showed that there were viable cells even when the optical density was low or no longer detectable and there was no plate count value. According to our results, flow cytometry is a suitable method for assessing the viability of bacteria in water samples. Furthermore, it permits fast detection of bacteria that are in a viable but nonculturable state, which are not detectable by conventional methods.