Peter L. Irwin

A 6×6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli

A protocol was developed using 96-well plates and multichannel pipettes for serial dilutions, followed by drop plating on agar in a 6 x 6 format. This protocol permits simultaneous plating of six dilutions which greatly decreases the number of plates utilized thereby saving incubator space for organisms such as Campylobacter which require unique environmental conditions.

1-Naphthyl phosphate as an enzymatic substrate for enzyme-linked immunomagnetic electrochemistry☆

Abstract We demonstrate substitution of the custom-synthesized alkaline phosphatase (AP) substrate, p -aminophenyl phosphate (pAPP), with the commercially available 1-naphthyl phosphate (1-NP) as applied in the enzyme-linked immunomagnetic electrochemical (ELIME) detection of the pathogenic bacterium, Escherichia coli O157:H7. ELIME entails ‘sandwiching’ bacterial analyte between antibody-coated magnetic beads and an AP-conjugated antibody. The beads (with or without bound bacteria) were localized onto the surface of magnetized graphite ink electrodes in a multi-well plate format. Enzyme substrate (pAPP or 1-NP) was added and conversion to an electroactive product was measured using Osteryoung square wave voltammetry. Using this technique, quantitative detection of E. coli O157:H7 bacterial cells was achieved with a minimum detectable level of ≤4.7×10 3 cells ml −1 in buffer or porcine carcass wash water within ca. 80 min.

Binding geometry, stoichiometry, and thermodynamics of cyclomalto-oligosaccharide (cyclodextrin) inclusion complex formation with chlorogenic acid, the major substrate of apple polyphenol oxidase

The inclusion complexes of cyclomaltohexaose (alpha-CD), cyclomaltoheptaose (beta-CD), cyclomaltooctaose (gamma-CD), and polymerized beta-CD (beta-CDn) with chlorogenic acid (CA), the major substrate of apple fruit polyphenol oxidase (PPO), were studied with regard to pH, ionic strength, and temperature in model buffer systems and apple juice. The thermodynamics of CD.CA inclusion complex formation, which were studied in solution using UV spectrophotometry, displayed enthalpy-entropy compensation typical of processes driven by solvation phenomena. We also found that the apparent association constants (K) of the CD.CA equilibrium were relatively insensitive to pH for beta-CD, compared to alpha- and gamma-CDs, but were subject to substantial enhancement at low ionic strengths. The beta-CD.CA inclusion complex was also characterized for binding geometry and stoichiometry at 9.4 T and 25 degrees C in 0.05 M Na phosphate buffer by 1H NMR spectroscopy. A 1:1 stoichiometric ratio for the complex was found using the method of continuous variations. 1H Spin-lattice relaxation and chemical-shift data indicate that the phenolic ring of CA docks within the cavity of beta-CD. The Ks for beta-, alpha-, and gamma-CD determined in apple juice, which contains a mixture of PPO substrates, were found to correlate with PPO activity-related data. Apple juice, treated with beta-CDn, did not brown until CA was added back. These latter findings strongly argue that the mechanism for inhibition of juice browning with cyclodextrins was mainly due to the binding of PPO substrates and not some other means such as enzyme inactivation via sequestration of Cu2+ by CDs.

Detection of Escherichia coli O157:H7 using immunomagnetic capture and luciferin-luciferase ATP measurement.

Abstract A procedure for rapid assay (about 30 min) of viable Escherichia coli O157:H7 in solutions is described. The bacteria, captured by specific immunomagnetic beads, were lysed by commercially available reagents. The released cellular ATP was determined by the chemiluminescence method using firefly lantern extracts. The ATP content of the E. coli capable of consuming oxygen was influenced by the presence of glucose. The immunomagnetic procedure was applied to determine the presence of E. coli O157:H7 in beef hamburger spiked with the bacteria. After a 6-h enrichment at 37°C, significant ATP luciferin-luciferase bioluminescence was detected from beef hamburger suspensions spiked with less than one CFU/ml of the E. coli . Possible utilization of the developed procedure for practical application is discussed.

Isolation of oligogalacturonic acids in gram quantities by preparative h.p.l.c.

Abstract Oligogalacturonic acids up to a degree of polymerization of 7 (d.p. 7) were isolated in gram quantities by preparative h.p.l.c. from endo-polygalacturonase- and pectate lyase-depolymerized polygalacturonic acid. A Dynamax-60A NH 2 (21.4 × 250 mm) 1-aminopropyl silica gel column was used with an isocratic acetate buffer (ca. 0.9 m , pH 5) mobile phase. Automated operation of the preparative h.p.l.c. system allowed for rapid, high-resolution separation and collection of oligogalacturonic acids that typically were 95–99% pure on a chromatographic peak area basis. The chromatographic system described represents an advance in oligogalacturonic acid isolation and purification methodology since it is faster, less labor intensive, and it provides higher isolation rates (over 300 mg/h of total oligosaccharides) than the traditionally used ambient pressure strong anion-exchange chromatography.

Antimicrobial activity of spherical silver nanoparticles prepared using a biocompatible macromolecular capping agent: evidence for induction of a greatly prolonged bacterial lag phase

BackgroundWe have evaluated the antimicrobial properties of Ag-based nanoparticles (Np s) using two solid phase bioassays and found that 10-20 μL of 0.3-3 μM keratin-stabilized Np s (depending on the starting bacterial concentration = CI) completely inhibited the growth of an equivalent volume of ca. 103 to 104 colony forming units per mL (CFU mL-1) Staphylococcus aureus, Salmonella Typhimurium, or Escherichia coli O157:H7 on solid surfaces. Even after one week at 37°C on solid media, no growth was observed. At lower Np concentrations (= [Np]s), visible colonies were observed but they eventually ceased growing.ResultsTo further study the physiology of this growth inhibition, we repeated these experiments in liquid phase by observing microbial growth via optical density at 590 nm (OD) at 37°C in the presence of a [Np] = 0 to 10-6 M. To extract various growth parameters we fit all OD[t] data to a common sigmoidal function which provides measures of the beginning and final OD values, a first-order rate constant (k), as well as the time to calculated 1/2-maximal OD (tm) which is a function of CI, k, as well as the microbiological lag time (T).Performing such experiments using a 96-well microtitre plate reader, we found that growth always occurred in solution but tm varied between 7 (controls; CI = 8 × 103 CFU mL-1) and > 20 hrs using either the citrate-([Np] ~ 3 × 10-7 M) or keratin-based ([Np] ~ 10-6 M) Np s and observed that {∂tm/∂ [Np]}citrate ~ 5 × 107 and {∂tm/∂ [Np]}keratin ~ 107 hr·L mol-1. We also found that there was little effect of Np s on S. aureus growth rates which varied only between k = 1.0 and 1.2 hr-1 (1.1 ± 0.075 hr-1). To test the idea that the Np s were changing the initial concentration (CI) of bacteria (i.e., cell death), we performed probabilistic calculations assuming that the perturbations in tm were due to CI alone. We found that such large perturbations in tm could only come about at a CI where the probability of any growth at all was small. This result indicates that much of the Np-induced change in tm was due to a greatly increased T (e.g., from ca. 1 to 15-20 hrs). For the solid phase assays we hypothesize that the bacteria eventually became non-culturable since they were inhibited from undergoing further cell division (T > many days).ConclusionWe propose that the difference between the solid and liquid system relates to the obvious difference in the exposure, or residence, time of the Np s with respect to the bacterial cell membrane inasmuch as when small, Np-inhibited colonies were selected and streaked on fresh (i.e., no Np s present) media, growth proceeded normally: e.g., a small, growth-inhibited colony resulted in a plateful of typical S. aureus colonies when streaked on fresh, solid media.

Ripening-related perturbations in apple cell wall nuclear spin dynamics

Abstract Spin-lattice ( 1 H T 1 , 23 Na + T 1 ) and rotating frame spin-lattice ( 1 H T 1 p , 13 C T 1 p ) relaxation times were measured on intact, critical point dried apple tissue at various degrees of ripeness using cross polarization and magic angle spinning (CPMAS) NMR techniques. Solid state carbonyl (δ172) 13 C T 1 p and 23 Na + -carboxylate anion T 1 values, which are inversely proportional to carboxylate reorientation rates, decreased 15–19% during the time course study. Carbonyl resonance 1 H T 1 s diminished by 63% as the tissue softened; a maximal decline of 42% was also observed in the 1 H T 1 s of nonspecific carbohydrate ring carbon signals (δ74) indicating an increase in both acidic and neutral polymer motion. Treatment of the cell wall with polygalacturonase resulted in a significant decrease in both carbonyl and ring carbon 1 H T 1 s (57 and 42%, respectively) demonstrating the important structural function of polyuronides not only in the middle lamellae but also in the primary cell wall.

Keratin capped silver nanoparticles--synthesis and characterization of a nanomaterial with desirable handling properties.

We report for the first time the stabilization of silver nanoparticles in good yield, average diameter 3.5 nm, using wool keratin hydrolysates as stabilizers. The nanoparticles are extremely stable as a suspension and can be lyophilized into a powder and easily reconstituted in solvent with no change in spectral properties relative to the initial suspension. The nanoparticles interact with nitrogen and oxygen moieties of the keratin hydrolysates under the pH conditions used in the synthesis and appear to act as cross-linkers between adjacent chains. The product has excellent handling properties which we believe will make it a very attractive biocompatible coating/additive, providing prolonged antimicrobial efficacy to a wide variety of products such as textiles, plastics, paints, orthopedic devices and others.

High-performance liquid chromatographic separation of oligogalacturonic acids on a cyclomaltoheptaose (β-cyclodextrin) bonded-phase column☆

Abstract A cyclomaltoheptaose (β-cyclodextrin) bonded-phase HPLC column was used for the first time to separate acidic oligosaccharides. Oligogalacturonic acids up to a degree of polymerization (dp) of 7 were separated with a 50:50 acetonitrile-sodium phosphate-buffered (pH 5) mobile phase and up to dp 17 with an isocratic pH 5.0, sodium phosphate-buffered mobile phase. A sodium acetate gradient elution allowed for improved resolution of all oligogalacturonic acids, up to a dp value of at least 24. Although the stationary phase contained no cationic or readily ionizable groups, these separations appeared to be governed by a classical anion exchange-type mechanism. The β-cyclodextrin-bonded phase, which displayed exceptional stability over one year of use, is a useful alternative to silica gel- or organic polymer-based anion exchangers for HPLC of acidic carbohydrates.

Preparative chromatography of oligogalacturonic acids

Abstract A mixture of oligogalacturonic acids was prepared by partial enzymatic hydrolysis of citrus pectin-derived α- d -polygalacturonic acid. Both a commercial fungal “pectinase” preparation and a purified endo-polygalacturonase isolated from this source by chromatography on carboxymethyl Sephadex C-50 were used to catalyze the hydrolysis. Different product distributions resulted, but even when using pectinase there was no evidence for the formation of unsaturated products from activity of polygalacturonic acid lyase. Individual oligogalacturonic acids were isolated by step-gradient elution (sodium formate, pH 4.7) from the macroporous strong base anion-exchange resin AG MP-1 in the formate form. Pure oligomers to heptagalacturonic acid were isolated in a single run, including gram quantities of tri-, tetra-, and pentagalacturonic acid. The individual oligogalacturonic acids were characterized by fast-atom bombardment mass spectrometry in positive and negative modes.