Nessler Reaction Research Articles

Bioanalytical insight into the life of microbial populations: A chemical monitoring of ureolytic bacteria growth

In this publication an alternative approach to investigations of bacterial growth is proposed. Contrary to the conventional physical methods it is based on enzyme activity detection. The procedure for real-time and on-line monitoring of microbial ureolytic activity (applied as a model experimental biosystem) in the flow analysis format is presented. The developed fully-mechanized bioanalytical flow system is composed of solenoid micropumps and microvalves actuated by Arduino microcontroller. The photometric detection based on Nessler reaction is performed using dedicated flow-through optoelectronic detector made of paired light emitting diodes. The developed bioanalytical system allows discrete assaying of microbial urease in the wide range of activity up to 5.4 U mL−1 with detection limit below 0.44 U mL−1, a high sensitivity in the linear range of response (up to 200 mV U−1 mL and relatively high throughput (9 detection per hour). The proposed differential procedure of measurements (i.e. a difference between peaks register for sample with and without external addition of urea is treated as an analytical signal) allows elimination of interfering effects from substrate and products of biocatalysed reaction as well as other components of medium used for microbial growth. The developed bioanalytical system was successfully applied for the control of growth of urease-positive bacteria strains (Proteus vulgaris, Klebsiella pneumoniae and Paracoccus yeei) including examination of effects from various microbial cultivation conditions like temperature, composition of culture medium and amount of substrate required for induction of bacterial enzymatic activity. The developed bioanalytical flow system can be applied for metabolic activity-based estimation of parameters of lag and log phases of microbial growth as well as for detection of decline phase.

Thanatochemistry at the crime scene: a microfluidic paper-based device for ammonium analysis in the vitreous humor

Most of the on-site approaches for inferring of the post-mortem interval are still based on observative data from the direct body inspection, whereas, objective and quantitative analyses, such as potassium in the vitreous humor, are require laboratory instrumentation and skilled personnel. The present paper presents a simple and low cost analytical method suitable for use at the crime scene for inferring the time since death. The method uses a microfluidic paper-based device (μPAD) for the determination of ammonium in the vitreous humor (VH) based on the selective interaction between the ammonium and the Nessler's reagent. The color change was measured in terms of “RGB distance” by using a simple and free smartphone application. The optimized device showed a limit of detection of 0.4 mmol L−1, with between days precision less than 9.3% expressed as relative standard deviation, and accuracy between days from 94.5% to 104.5%. The selectivity of the Nessler's reaction was tested towards the main vitreous humor compounds, and no significant interferences were found. This paper-based analytical device was successfully used for the determination of ammonium ion in VH samples from forensic autopsies. The results obtained with the proposed method, although for a limited number of cases (n = 25), showed a close correlation with the data obtained with an instrumental analysis based on capillary electrophoresis. Moreover, in order to make the evaluation of results as simple as possible, a direct correlation between the color intensity, expressed as RGB distance, and the post-mortem interval was studied and a significant correlation was found (R2 > 0.78). In conclusion, the present preliminary study showes that the proposed device could be an additional tool to the traditional methods for a more accurate, although still presumptive, estimation of the time of death directly at the crime scene.

Deep eutectic solvents functionalized polymers for easily and efficiently promoting biocatalysis

Biocompatible and magnetic polymers of poly(vinyl pyrrolidone deep eutectic solvent, VP DES) were investigated to determine whether they could efficiently adsorb L-asparaginase, which is an important enzyme inhibiting the growth of certain tumor cells. A series of poly(VP DES)s were successfully obtained, and that based on VP–malonic acid (VP/MA molar ratio of 1:1) was found to be the best for L-asparaginase adsorption. In addition, L-asparaginase was easily separated with biocompatible poly(VP-MA DES) from Escherichia coli extracts under a magnetic field with a high specific activity toward the hydrolysis of L-asparagine, as determined by the Nessler reaction method. Based on this work, poly(VP-MA DES)s seem ideal carriers via adsorption for the delivery of specific enzyme for the biocatalysis.

A mechanized urease activity assay

Two fully mechanized flow analysis systems for urease activity assays have been developed, characterized and compared. Both of them are based on almost the same compact system of solenoid micropumps and microvalves controlled and actuated by highly effective, low-power and economic Arduino microcontroller. For photometric detection of ammonia formed in the course of enzymatic hydrolysis of urea, the Berthelot method and the Nessler reaction have been examined. For both these detection schemes very simple dedicated optoelectronic flow-through detectors made of paired light emitting diodes have been developed. In both systems single enzyme assay lasting a few minutes allows determination of urease in activity range 0.02–5.3 U mL−1 with detection limit 0.02 U mL−1 and in 1.3–5.3 U mL−1 range with 0.75 U mL−1 detection limit for Nessler reaction and Berthelot method based systems, respectively. When compared with mechanized Berthelot method, the bioanalytical system based on Nessler reaction offers higher sensitivity, lower detection/determination limits, better selectivity and lower cost of the assay. It has been demonstrated that the developed bioanalytical flow systems could be useful for urease determination in complex biological matrix like plant extracts and media for microbial cultures as well as for inhibitive determination of heavy metals at sub-ppm levels.

Bioavailable inorganic forms of nitrogen and phosphorus in extracts of herbs, flowers and bark of medicinal plants

The concentrations of total nitrogen and phosphorus, and their water-extractable inorganic chemical forms – nitrate nitrogen and – phosphate phosphorus, were analyzed in a set of 58 samples of Polish medicinal plant materials: herbs, flowers and bark. Total and extractable inorganic phosphorus were determined spectrophotometrically by the molybdenum blue method, the total nitrogen was based on Nessler reaction, and the nitrate nitrogen on the nitration method. On a dry weight basis, the total nitrogen covered the values from 4.69 to 27.42 mg/g in herbs, from 11.10 to 40.67 mg/g in flowers and from 5.92 to 9.77 mg/g in bark samples. Total phosphorus was found from 2.70 to 10.19 mg/g in herbs, from 2.15 to 8.82 mg/g in flowers and from 0.46 to 1.30 mg/g in bark samples. On average, the nitrate nitrogen represented the fraction from 2.6% (flowers) to 15.1% (bark) of the total amount of nitrogen, whereas inorganic phosphate phosphorus was from 28.3% (herbs) to 60.2% (bark) of the total amount of this element. Correlation analysis revealed that the total N is not correlated with N–NO3 except the bark samples, but P–PO4 was positively related to the total P level, with the exception of herbs (a<0.05). Student’s t-test confirmed a statistically significant difference between the level of N, P and their water-extractable inorganic forms determined in bark with the comparison with the values found in herbs and flowers. Potentially bioavailable inorganic forms of nitrogen and phosphorus were compared with the nutritional norms of WHO/FAO and RDA, indicating that they can deliver significant amounts of N and P to humans.

A semi-automated method for the determination of ammonia in urine using the Nessler reaction.

Species in urine which were likely to interfere with the determination of ammonia by Nesslerisation were adsorbed onto a precipitate of zinc hydroxide formed in situ in each sample. The ammonia concentrations in the subsequent supernatant solutions were measured using the Nessler method, adapted for use on a continuous flow analysis system. These measurements were made against a series of standards prepared from Analar grade ammonium chloride. The accuracy, reproducibility, and sensitivity of this adaptation were examined and were found to be within acceptable limits.

Automated determination of bacterial asparaginase and glutaminase

A method is described for the direct determination of l-asparaginase and l-glutaminase in bacterial suspensions using an enzyme assay based upon the Nessler reaction and a protein determination based upon the Folin reaction operating in tandem. The amidase assay operates over the range 0.005–0.04 IU/ml and the protein determination over the range 0.005–0.04 mg/ml, permitting the determination of specific activities over the range 0.125–8 IU/mg.

Gravimetric determination of ammonium ion by Nessler's reagent

The brownish precipitate in Nessler's reaction was used for the gravimetric determination of ammonium ion in an aqueous solution. The composition of the precipitate is NHg2I, and the gravimetric factor of nitrogen is very small (0.02584).The recommended procedure are followings; The two-fold equivalent amount of Nessler's reagent (HgI42- : 2×10-2F, OH- : 4×10-1F ) is added slowly to 25 ml of sample solution. It is allowed to stand for at least ten hours at room temperature and then warmed for three hours on steam bath. The digested precipitate is filtered through a fine-pored glass filter (average pore size : 1 μ), washed by deionized water and dried at 150°C in air.Ammonium ion ranging from 2 to 4 mg as nitrogen in 25 ml of ammonium chloride solution was determined within error of 2.1% and standard deviation of 6.8%. Negative errors were observed for the lower contents of the ion, and also significant positive errors for the higher contents.The recognized interfering substances are comlexing agents such as potassium cyanide, potassium thiocyanide, ethylenediamine and pyridine, and reducing agents such as hydroxylamine hydrochloride and hydrazine hydrochloride.

Determination of the Partial Pressure of Ammonia in Soil Air

AbstractA method for determining partial pressures of NH3 from 0.01 to 0.50 mm Hg in soil air was studied. Soil samples which had been treated with various amounts of ammonium hydroxide were placed in 1‐liter polyethylene bottles and allowed to equilibrate. A known volume of air from the bottle was removed by partially compressing the bottle with a press. The air was bubbled through 0.1N HCl and the ammonium content of the acid determined by the Nessler reaction. The partial pressure of NH3 (PNH3) was calculated from the ideal gas law equation.The method gives a reliable estimate of the partial pressure of NH3 in soil air. It was shown that the PNH3 of a sample placed in the polyethylene bottle could be determined after a 1‐hour equilibration period.Samples which were sealed in the polyethylene bottles for 48 hours had lower PNH3 and higher PCO2 values than samples placed in the bottles for only 1 hour. Reduced PNH values in bottles which had been sealed for 1 day or more were attributed to pH reduction due to CO2 accumulation.Germination of corn seeds (Zea mays L.) was damaged when the initial PNH2 value was 0.156 and final values were between 0.077 and 0.104 mm Hg. The germination of corn seeds was not reduced when initial PNH3 values were 0.091 and final values were 0.067 and 0.048 mm Hg.

Quantitative Range of Nessler's Reaction with Ammonia

Abstract The minimum concentration of mercury required for Nessler's reaction with ammonia approaches 86% of the stoichiometric value. When the mercury concentration is low, nitrogen can be determined in small amounts in the ultraviolet region. The maximum accuracy and working range are shown for 350-600 mµ.

The basis of Nessler's reaction in clinical chemistry and its use for urea estimations

Estimation of ammonium ions in biological fluids was investigated within a concentration of 0.008–0.036 mg NH4+. Nessler's equilibrium of the reaction is displaced towards the right (the side of the coloured complex) by increasing the concentration of KOH and by increasing the temperature. It occurs more rapidly at a higher temperature. Reproducible results can be obtained with a set KOH-concentration, time and temperature, i.e. using the same KOH-concentration, the same time and the same temperature. It was shown that the reaction is suitable for the estimation of urea after enzymic hydrolysis.

New Nessler reagent and its use in the direct nesslerisation of Kjeldahl digests

AbstractThe instability of conventional Nessler reagents is shown to be the cause of inaccuracy when they are used to estimate ammonia in solution. A critical investigation of the Nessler reaction has resulted in the formulation of a satisfactory, aqueous reagent. Use of this reagent along with gum arabic, which protects the ammonia‐colour‐complex from flocculation, has permitted the problem of interference to be clarified and by incorporating a method for controlling interference a procedure has been worked out for directly nesslerising Kjeldahl digests. When applied to the determination of the nitrogen content of plant material, the procedure is comparable in accuracy and precision to that of a distillation‐titration method. The reagent is sensitive to 5 μg. of ammoniacal nitrogen in solution and the precision of the method is about 1·4%.

魚肉アンモニヤの簡易比色測定法

Some experiments were carried out to apply Nessler's colorimetric method as a simple device for measuring the quantities of ammonia in fish meat. It was found that the colour reaction by Nessler's reagent for extracts of fish meat, does not indicate characteristic colour which is to be induced by ammonia, but colours differently; and it was shown that some substances which obstruct Nessler's reaction are removed by adding the adsorbent which does not influence ammonia in acidified solutions. Therefore, Nessler's method may be adapted as a simpiem method for the estimation of ammonia in fish meat, by the following items: 1) treating the aqueous extracts of fish meat with protein-precipitants charged with definite acidity and adsorbent, 2) changing, alkali earth metal ions into complex ions, dissolved out to filtrate from adsorbent, 3) preparing a durable standard solution.

Purification of Paralytic Shellfish Poison by Filtration Through Active Charcoal

SummaryA simple and efficient method for the purification of shellfish poison consists in differential retardation when the poison is filtered through a charcoal column. The resulting products are low in ash, have lost their fluorescence, have an equal number of Cl and N atoms and give a characteristic Nessler reaction.

Photometric Investigation of Nessler Reaction and Witting Method for Determination of Ammonia in Sea Water

Photometric Investigation of Nessler Reaction and Witting Method for Determination of Ammonia in Sea Water