Tadayuki Tsukatani

Comparison of the WST-8 colorimetric method and the CLSI broth microdilution method for susceptibility testing against drug-resistant bacteria.

The minimum inhibitory concentrations (MICs) obtained from the susceptibility testing of various bacteria to antibiotics were determined by a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone as an electron mediator and compared with those obtained by the broth microdilution methods approved by the Clinical and Laboratory Standard Institute (CLSI). Especially for drug-resistant bacteria, the CLSI method at an incubation time of 24h tended to give lower MICs. The extension of incubation time was necessary to obtain consistent MICs for drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococi (VRE) and multi-drug resistant Pseudomonas aeruginosa (MDRP) in the broth microdilution method. There was excellent agreement between the MICs determined after 24h using the WST-8 colorimetric method and those obtained after 48-96 h using the broth microdilution method. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of consistent MICs for drug-resistant bacteria.

Colorimetric cell proliferation assay for microorganisms in microtiter plate using water-soluble tetrazolium salts.

A colorimetric method to assay cell proliferation of microorganisms in 96-well microtiter plates using water-soluble tetrazolium salts and electron mediators was developed. Combinations of 6 kinds of water-soluble tetrazolium salts and 27 kinds of electron mediators that considered the metabolic efficiency of microorganisms and the influence with medium components were investigated. 2-Methyl-1,4-naphthoquinone (NQ) was reduced most effectively by various species of microorganisms, and a combination of WST-8 as a water-soluble tetrazolium salt with 2-methyl-1,4-NQ repressed the increase in background due to medium components. In the presence of 2-methyl-1,4-NQ, WST-8 was reduced by microbial cells to formazan, which exhibited maximum absorbance at 460 nm. The proposed tetrazolium method could be applied to measure proliferations of various microbial cells including 3 kinds of yeast, 9 kinds of Gram-positive bacteria, and 10 kinds of Gram-negative bacteria. Linear relationships between the absorbance and viable microbial cell density were obtained in all microorganisms, suggesting that the absorbance change reflected the microbial cell proliferation.

Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances.

The applicability of a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antimicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antimicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.

Simultaneous quantitation of citrate and isocitrate in citrus juice by a flow-injection method based on the use of enzyme reactors

A method for the simultaneous quantitation of citrate and isocitrate in citrus juice by flow-injection analysis (FIA) was developed using two enzyme reactors in parallel and an electrochemical flow-through cell. Citrate was quantitated with a co-immobilized citrate lyase (CL) and oxaloacetate decarboxylase (OD) reactor and an immobilized pyruvate oxidase (PO) reactor as the upstream and downstream reactors, respectively. Hydrogen peroxide produced by the CL·OD and PO reactors was monitored amperometrically with a platinum electrode. Since CL is subjected to end-product inhibition, we tried to increase the tolerated frequency of injections of the CL · OD reactor by lowering the amount of citrate in the reaction solution. The response at the 0.05 mM (2.5 nmol) and 0.10 mM (5.0 nmol) level remained steady for 196 and 116 injections, respectively. Isocitrate was quantitated by monitoring the NADPH formed by NADP-specific isocitrate dehydrogenase (NADP-ICDH) or by measuring the hydrogen peroxide produced in the coupled reactions catalyzed by NAD+-specific isocitrate dehydrogenase (NAD-ICDH) and NADH oxidase (NOD). Since citrate interfered with the signal for isocitrate alone in the case of the NAD-ICDH · NOD reactor, an NADP-ICDH reactor was used for the quantitation of isocitrate in citrus juice. The volume of the sample loop in the citrate line was decreased from 50 μl to 1 μl for the simultaneous quantitation of citrate and isocitrate. Linear relationships between the responses and the concentrations of isocitrate and citrate were observed in the ranges 2.5–50 μM and 0.25–5.00 mM, respectively, i.e. over a range of 0.25–5.00 nmol in the reaction solution. The relative standard deviations for ten successive injections were less than 2% for 5.0 mM citrate and for 50 μM isocitrate. The system was applied to the determination of citrate and isocitrate in citrus juice. The results were in good agreement with those obtained by a conventional method (F-kit method), suggesting that this system might be applicable for the detection of the adulteration of citrus products.

Sequential fluorometric quantification of malic acid enantiomers by a single line flow-injection system using immobilized-enzyme reactors

A method for the sequential enantiomeric quantification of d-malate and l-malate by a single line flow-injection analysis was developed using immobilized-enzyme reactors and fluorescence detection. An immobilized d-malate dehydrogenase (d-MDH) reactor and an immobilized l-malate dehydrogenase (l-MDH) reactor were introduced into the flow line in series. Sample and coenzyme (NAD(+) or NADP(+)) were injected into the flow line by an open sandwich method. d-Malate was selectively oxidized by d-MDH when NAD(+) was injected with a sample. When NADP(+) was injected with a sample, l-malate was oxidized only by l-MDH. NADH or NADPH produced by the immobilized-enzyme reactors was monitored fluorometrically at 455nm (excitation at 340nm). Linear relationships between the responses and concentrations of d-malate and l-malate were observed in the ranges of 1 x 10(-6)-1 x 10(-4)M and 1 x 10(-6)-2 x 10(-4)M, respectively. The relative standard deviations for ten successive injections were less than 2% at the 0.1mM level. This analytical method was applied to the sequential quantification of d-malate and l-malate in fruit juices and soft drinks, and the results showed good agreement with those obtained using conventional method (F-kit method).

Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts.

A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B(6), biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B(6) in various foodstuffs. There was good agreement between vitamin B(6) concentrations determined after 24h using the WST-8 colorimetric method and those obtained after 48h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate.

Measurement of L-Malate Using Immobilized Enzyme Reactors: Comparison of Results Obtained with Four Different Enzymatic Systems.

For the measurement of malate by an enzyme sensor, we did a comparative study using malate dehydrogenase (MDH) alone, MDH and glutamate oxaloacetate transaminase (GOT) together, a malic enzyme (ME) that requires NADP as a cofactor, and MDH and NADH oxidase together. With respect to the response of each reactor to 0.5 mM L-malate, the systems using ME alone and MDH plus NADH oxidase gave high values. The ranges of measurements were 0.05-1.00 mM (MDH alone), 0.01-0.05 mM (MDH plus GOT), 0.01-0.50 mM (ME alone) and 0.02-1.00 mM (MDH plus NADH oxidase). In the system with MDH alone, however, reducing sugars in the sample interfered with measurements and it was impossible to use this system for practical analysis of fruit samples. By contrast, the systems using ME alone or MDH plus NADH oxidase were unaffected by the presence of reducing sugars and were suitable for analysis of samples. Thus, the MDH-NADH oxidase system is recommended for practical analyses of samples.

New tetrazolium method for phosphatase assay using ascorbic acid 2-phosphate as a substrate.

A new method to assay alkaline and acid phosphatases assay using ascorbic acid 2-phosphate (AsA-P) and ditetrazolium salt nitroblue tetrazolium chloride (NBT) was developed. AsA-P is hydrolyzed in the presence of phosphatase to yield ascorbic acid. In turn, the ascorbic acid reduces NBT directly or indirectly, opening the tetrazole ring to produce an insoluble formazan as a colored precipitate. The proposed method for alkaline phosphatase was compared with a conventional method in which 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used in combination with NBT in the dot blots of a dilution series of beta-lactoglobulin. AsA-P reduced NBT more effectively than BCIP in the presence of alkaline phosphatase. AsA-P could be also used as the chromogenic substrate for an acid phosphatase assay in the presence of phenazinium methylsulfate and NBT.

Enzymatic Quantification of L-Tartrate in Wines and Grapes by Using the Secondary Activity of D-Malate Dehydrogenase.

L-Tartrate in wines and grapes was enzymatically quantified by using the secondary activity of D-malate dehydrogenase (D-MDH). NADH formed by the D-MDH reaction was monitored spectrophotometrically. Under the optimal conditions, L-tartrate (a 1.0 mM sample solution) was fully oxidized by D-MDH in 30 min. A linear relationship was obtained between the absorbance difference and the L-tartrate concentration in the range of a 0.02-1.0 mM sample solution with a correlation coefficient of 0.9991. The relative standard deviation from ten measurements was 1.71% at the 1.0 mM sample solution level. The proposed method was compared with HPLC, and the values determined by both methods were in good agreement.

Metabolism of Benzoquinone by Yeast Cells and Oxidative Characteristics of Corresponding Hydroquinone: Application to Highly Sensitive Measurement of Yeast Cell Density by Using Benzoquinone and a Chemiluminescent Probe

The metabolic efficiency of seven derivatives of 1,4-benzoquinone (BQ) by yeast cells and the oxidative characteristics of the corresponding hydroquinones (HQs) were studied by electrochemical, spectrophotometric and chemiluminescent methods. The spectrophotometric method was based on the reduction of a tetrazolium salt to formazan dye during the autoxidation of HQs generated by yeast cells under alkaline conditions. The amounts of HQs detected directly by the electrochemical method did not agree with those calculated from the formazan dye obtained by the spectrophotometric method. A tetrazolium salt was reduced to a formazan dye by both the superoxide anion radical (O2−•) generated during the autoxidation of 2,3,5,6-tetramethyl-1,4-HQ and by HQ itself. Little formazan dye was formed, and hydrogen peroxide (H2O2) was then finally produced during the autoxidation of 1,4-HQ or 2-methyl-1,4-HQ. Formazan dye and H2O2 were generated at a certain ratio during the autoxidation of derivatives of dimethyl-1,4-HQ or 2,3,5-trimethyl-1,4-HQ. The analytical method based on chemiluminescence with lucigenin and 2,3,5,6-tetramethyl-1,4-BQ was applied to highly sensitive measurement of the yeast cell density. A linear relationship between the chemiluminescence intensity and viable cell density was obtained in the range of 1.2×103–4.8×104 cells/ml. The detection limit was 4.8×102 cells/ml.