An Efficient Method for Qualitative Screening of Phosphate-Solubilizing Bacteria

An efficient protocol was developed for qualitative screening of phosphate-solubilizing bacteria, based upon visual observation. Our results indicate that, by using our formulation containing bromophenol blue, it is possible to quickly screen on a qualitative basis the phosphate-solubilizing bacteria. Qualitative analysis of the phosphate solubilized by various groups correlated well with grouping based upon quantitative analysis of bacteria isolated from soil, effect of carbon, nitrogen, salts, and phosphate solubilization-defective transposon mutants. However, unlike quantitative analysis methods that involve time-consuming biochemical procedures, the time for screening phosphate-solubilizing bacteria is significantly reduced by using our simple protocol. Therefore, it is envisaged that usage of this formulation based upon qualitative analysis will be salutary for the quick screening of phosphate-solubilizing bacteria. Our results indicate that the formulation can also be used as a quality control test for expeditiously screening the commercial bioinoculant preparations, based on phosphate solubilizers. Received: 17 November 2000 / Accepted: 22 December 2000     

Influence of Environmental Temperature on Biocalcification by Non-sporing Freshwater Bacteria

The process of biocalcification, recognised as precipitation of calcium carbonate, has been described as a widespread phenomenon associated with a wide range of different bacterial species. This biocalcifying activity, and factors that affect it, have been widely studied in moderately halophilic bacteria but there is a lack of information on factors that affect biocalcification by freshwater bacteria. In this paper, we study how temperature can affect biocalcification by freshwater bacteria that potentially could be used for the process of bioconsolidation during conservation. Ten isolates were characterised by standard biochemical and API 20NE tests. Their biocalcifying activity was studied at temperatures between 10 and 40°C in B4 liquid medium. Mineralogical and quantitative analyses of the crystals were carried out by XRD, and morphological studies by SEM. Biocalcification only occurred when bacteria were present and were able to grow. Carbonate precipitation by bacteria increased with time and temperature of incubation. Temperature affected not only the amount of precipitation but also crystal quality and morphology. As bioconsolidant agents, these organisms could be applied to stone when the temperature does not exceed 40°C depending on the type of isolate.     

Model film studies in enzyme histochemistry with special reference to glucose-6-phosphate dehydrogenase

Summary This paper deals with the progress made over the last few years in our understanding of enzyme cytochemical staining methods as studied using a fundamental approach with the aid of a model system of thin gel films. Although model films with a matrix of polyacrylamide have been mostly used, the properties and possible applications of other matrices are also reviewed. The chemical aspects of the entrapment of enzyme molecules into a matrix are summarized. Special attention has been paid in model film studies to the principles of the trapping reaction of a diffusable precursor resulting from the enzymatic conversion of a substrate. They are considered here as they concern the cytochemical demonstration of acid phosphatase activity with a lead salt. The effect of fixatives on different enzyme activities, the diffusion rate of substrates and chromogenic compounds to the enzyme site, and enzyme kinetics under cytochemical conditions are also discussed, since they are factors which influence the final results of the staining procedures. The advantage of model film studies in enabling the direct correlation of cytochemical and biochemical results is outlined with special reference to the cytochemical determination of glucose-6-phosphate dehydrogenase with Tetra Nitro BT. A method for determining enzyme activities in the soluble fraction of isolated cells after incorporation in model films is described for the first time. This method has proved to be highly appropriate for microscopical observations of glucose-6-phosphate dehydrogenase activity in single cells, because it results in a good morphology and no formazan precipitaties outside the cells. On the other hand, this type of model film forms a bridge between fundamental model film studies using purified enzyme and quantitative enzyme cytochemistry performedin situ.     

Lysozyme for capture of microorganisms on protein biochips

Lysozyme placed on the SiO₂ surfaces that have previously been derivatized with C₁₈ coating will capture both Escherichia coli and Listeria monocytogenes cells from PBS buffer at pH 7.2. This phenomenon is of significance for the design and fabrication of protein biochips that are designed to capture bacteria from buffer or water so that these can be further interrogated with respect to possible pathogenicity. Fluorescent microscopy shows that two types of bacteria (gram-negative E. coli and gram-positive Listeria spp.) will be adsorbed by lysozyme placed on the surface of the biochip but that strong adsorption of the bacteria is reduced but not eliminated when Tween 20 is present (at 0.5%) in the PBS buffer in which the cells are suspended. In comparison, Tween 20 and Bovine Serum Albumin (BSA) almost completely block adsorption of these bacteria on C₁₈ coated surfaces. The combination of a lysozyme surface with Tween 20 gives a greater degree of adsorption of L. monocytogenes than E. coli, and hence suggests selectivity for the more hydrophobic E. coli may be reduced by the Tween 20. This paper presents protocols for preparing protein-coated, SiO₂ surfaces and the effect of buffer containing Tween 20 on adsorption of bacteria by SiO₂ surfaces coated with C₁₈ to which BSA, lysozyme or C11E9 antibody is immobilized at pH 7.2 and ambient temperature.     

Quantitative Brewster angle microscopy of the surface film of human broncho-alveolar lavage fluid

The morphology, thickness and surface pressure of the surfactant film of broncho-alveoalar lavage (BAL) fluid from patients with sarcoidosis were investigated during spontaneous adsorption of the BAL's surface active material at the air/aqueous buffer interface at 37 degrees C. The biochemical parameters of the BAL fluid determined were protein (Lowry), total phospholipids (from phosphate after ashing) and the individual phospholipids (HPLC). During the spontaneous adsorption of the pulmonary surfactant the surface pressure increased from initially 26 mN/m to 44 mN/m in the equilibrium state. Simultaneously to the increase of the surface pressure, a continuous increase of the reflectivity signal was observed by quantitative Brewster angle microscopy (BAM). The film thickness is calculated from the reflectivity values using an optical model. The effect of the uncertainty of the refractive index, which has to be estimated, is discussed. The BAM images show the inhomogeneous nature of the surfactant film with three distinct phases of different reflectivity, even at relatively low surface pressures. For the brightest phase, the thickness amounts to approximately 12 nm in the equilibrium state of adsorption. This suggests a multilamellar structure. Additionally, we found visual evidence for an adsorption mechanism involving the spreading of vesicles at the interface, in agreement with published results. Differences in the morphology and thickness of the pulmonary surfactant film reported in the literature are obviously due to the varying experimental conditions and materials. We think that the experimental conditions chosen in our study provide a more realistic view of the structure in the lungs in vivo.     

Nanocomposite of functionalized multiwall carbon nanotubes with nafion, nano platinum, and nano gold biosensing film for simultaneous determination of ascorbic acid, epinephrine, and uric acid

A unique bimetallic, nano platinum (Pt) with nano gold (Au) on nafion (NF) incorporated with functionalized multiwall carbon nanotubes (f-MWCNTs) composite film (f-MWCNTs-NF-PtAu) was developed by the potentiostatic method. The composite film exhibits promising efficient catalytic activity towards the oxidation of mixture of biochemical compounds and simultaneous measurement of ascorbate anion, epinephrine and urate anion in aqueous buffer solution (pH 6.75). Both, the cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used for the measurement of electroanalytical properties of neurotransmitters by means of composite film modified electrodes. Well-separated voltammetric peaks were obtained for ascorbate, epinephrine and urate anions with the peak separations of 0.222 and 0.131V. The composite film can also be produced on gold and transparent semiconductor indium tin oxide electrodes for different kinds of studies such as electrochemical quartz crystal microbalance (EQCM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The incorporation of Pt and Au onto the f-MWCNTs-NF was revealed by the EQCM technique and the morphology of the film was studied using SEM, AFM and scanning electrochemical microscopy (SECM) techniques. Further, extensive studies were carried out using SECM for obtaining the surface current topographic images of composite film modified electrodes, and these indicated the presence of f-MWCNTs-NF-PtAu composite film on the electrode.     

Binding of Tris to Bacillus licheniformis alpha-amylase can affect its starch hydrolysis activity

Bacillus licheniformis alpha-amylase (BLA) is routinely used as a model thermostable amylase in biochemical studies. Its starch hydrolysis activity has recently been studied in Tris buffer. Here, we address the question that whether the application of Tris buffer may influence the results of BLA activity analyses. Based on the inhibition studies and docking simulations, we suggest that Tris molecule is a competitive inhibitor of starch-hydrolyzing activity of BLA, and it has a high tendency to bind the enzyme active site. Hence, it is critically important to consider such effect when interpreting the results of activity studies of this enzyme in Tris buffer.     

Vacuum Deposited Triple‐Cation Mixed‐Halide Perovskite Solar Cells

Hybrid lead halide perovskites are promising materials for future photovoltaics applications. Their spectral response can be readily tuned by controlling the halide composition, while their stability is strongly dependent on the film morphology and on the type of organic cation used. Mixed cation and mixed halide systems have led to the most efficient and stable perovskite solar cells reported, so far they are prepared exclusively by solution‐processing. This might be due to the technical difficulties associated with the vacuum deposition from multiple thermal sources, requiring a high level of control over the deposition rate of each precursor during the film formation. In this report, thermal vacuum deposition with multiple sources (3 and 4) is used to prepare for the first time, multications/anions perovskite compounds. These thin‐film absorbers are implemented into fully vacuum deposited solar cells using doped organic semiconductors. A maximum power conversion efficiency of 16% is obtained, with promising device stability. The importance of the control over the film morphology is highlighted, which differs substantially when these compounds are vacuum processed. Avenues to improve the morphology and hence the performance of fully vacuum processed multications/anions perovskite solar cells are proposed.     

An agarose slide method to follow the fate of bacteria within digestive vacuoles of protozoa

Abstract A method to follow the fate of ingested bacteria within digestive vacuoles of protozoa is presented. Tetrahymena pyriformis , previously fed with bacteria, is deposited onto glass microscope slides covered with a film of nutritive agarose. The protozoa lyse and the digestive vacuoles containing the bacteria stay undamaged and can be observed microscopically. After incubation, microcolonies reveal those vacuoles which contained living bacteria. The method can be used to study the survival ability of the ingested bacteria. It is a potentially valuable technique for studies on digestion efficacy, virulence ability, or escape mechanisms of bacteria from digestion.     

Effect of buffer on kinetics of proton equilibration with a protonable group

The laser-induced proton pulse generates a massive, brief, proton pulse capable of perturbing biochemical equilibria. The time resolution of the monitoring system can follow the diffusion-controlled protonation of specific sites on macromolecular bodies [Gutman, M. (1984) Methods Biochem. Anal. 30, 1-103]. In order to apply this method in enzymology, one must first evaluate how the buffer capacity of biochemical systems (substrates and proteins) will affect the observed dynamics. Unlike equilibrium measurements, where buffer is an inert component, in kinetic studies buffer modulates the observed dynamics. In this paper we analyze the effect of buffer on the dynamics of protonation in a model system. We describe the experimental technique and introduce the mathematical formalism that determines the various rate constants involved in the reaction. The analysis of the experiments indicates that in buffered solution proton flux is carried by two mechanisms: (A) proton dissociation followed by free proton diffusion; (B) collisional proton transfer between small diffusing solutes. We demonstrate how to evaluate the contribution of each pathway to the overall proton flux.     

Methods for quantitative determination of ethylene in aqueous solutions for biological studies

Abstract. In many plant physiological studies, quantitative determination of ethylene in aqueous solutions is necessary. We describe two methods for such measurements based on vacuum extraction, or on stripping ethylene from solution with the carrier gas of a gas chromatograph. The gas-stripping technique is limited by the small sample volumes that can be used, whereas the vacuum extraction technique can accommodate much larger volumes and is useful when the ethylene concentration in the solution is very low. The use of these techniques will facilitate the treatment of submerged plant tissues with known amounts of ethylene, and also the quantitative determination of ethylene from tissues grown in either static or continuous flow liquid cultures.     

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.     

地膜降解途径及机理研究进展

我国地膜使用量已占农业塑料薄膜使用量的半数以上,其大规模使用在带来巨大经济价值的同时也造成了“白色污染”,残膜难回收、难降解,直接影响土壤的再生能力。本文综述了我国地膜使用现状及其降解方面的研究进展,分别对地膜降解的生物与非生物途径和机理进行了概括,重点分析了非生物降解途径中光催化、金属离子掺杂等对聚烯烃降解的协同效应,以及生物降解途径中不同来源的菌和酶对聚烯烃降解效率的影响,并展望未来发展出更为高效的地膜降解方式,为后续地膜降解及环境中微塑料的降解研究提供参考。     

Effects of fixation and substrate protection on the isoenzymes of aspartate aminotransferase studied in a quantitative cytochemical model system

The cytochemical technique of Lee and Torack for the demonstration of aspartate aminotransferase activity was tested on a model system consisting of either total liver homogenate or the mitochondrial or soluble cytoplasmic fraction, incorporated in polyacrylamide film. After incubation of portions of film in a medium of α-ketoglutarate, L-aspartate, and lead nitrate, the lead oxaloacetate formed was converted to lead sulfide. The absorbance determined at 520 nm in a film spectrophotometer and expressed in terms of unit weight of film provided a measure of the contained enzymatic activity, and was directly proportional to the concentration of chemically determined oxaloacetate in the film. Both mitochondrial and "soluble" isozymes of aspartate aminotransferase reacted with the cytochemical media to a quantitatively similar degree, but were considerably inactivated after 15 min of treatment with 1% glutaraldehyde or 3.7% formaldehyde in imidazole buffer, the rate of inactivation being greater for the soluble isozyme. Application of the principle of substrate protection delayed inactivation. Thus, for both isozymes the rate of inactivation decreased if ketoglutarate was added to the fixative. Similarly, it was shown that the optimal incubation medium for the demonstration of the soluble isozyme must contain 4 mM of α-ketoglutarate and 20 mM of L-aspartate. Under these conditions the turnover-number for the cytochemical system is 70% of the value obtained from biochemical estimations. Cytochemical K_m values differed for each isozyme and were in accord with values determined by biochemical techniques, indicating that the model system can be used as a link between biochemical and cytochemical data in enzymatic studies.     

Degassing‐assisted patterning of cell culture surfaces

We developed an alternative patterning technique which is capable of producing both topographic and biochemical features for cell culture studies. This technique is based on microaspiration induced with a degassed poly (dimethylsiloxane) (PDMS) mold. After degassing in a rough vacuum chamber and placed on a sample surface, liquid solution can be aspired through channels and cavities created in the PDMS mold. Depending on the composition of the solution and the associated drying or incubation processes, a variety of surface patterns can be produced without applying external pressure. For demonstration, we fabricated agarose gel microwells and biomolecule patterns either on a glass plate or in a cell culture Petri dish, both applicable for cell culture studies. Biotechnol. Bioeng. 2010. 105: 854–859. © 2009 Wiley Periodicals, Inc.     

Kinetics of adsorption,desorption, and exchange of alpha-chymotrypsin and lysozyme on poly(ethyleneterephthalate) tracked film and track-etched membrane

Adsorption kinetics of (125)I-radiolabeled alpha-chymotrypsin at pH 8.6 was studied in a laminar regime between two walls of poly(ethyleneterephthalate) tracked films and membranes. Adsorption kinetics in the presence of solution (10 microg/mL), desorption by rinsing with buffer, and the following exchange of proteins by flowing unlabeled solution were measured. At pH 8.6, alpha-chymotrypsin is almost neutral and can be mostly removed from the film surface, contrary to positive lysozyme adsorbed at pH 7.4. Results suggest that alpha-chymotrypsin is irreversibly adsorbed in pores, while desorption and exchange occur on membrane flat faces. A method is proposed to determine adsorption kinetics in the pores. Kinetics of desorption and exchange of alpha-chymotrypsin from the film surface can be described by stretched exponential functions in the examined time domain with the same exponent, beta approximately 0.62, which does not depend also on the former adsorption duration. However, the mean residence time at the interface is about 2.5 times greater in the presence of only the buffer than that in the presence of solution. This effect could be explained by a fast exchange at the arrival of unlabeled solution for a part of the adsorbed population.     

Evaluation of a cup scrub technique for quantification of the microbial flora on bovine skin

A cup-scrub technique devised for sampling the human skin surface microflora was evaluated in cattle. Scrub samples from bovine skin contained clumps of squama and bacterial microcolonies which were progressively broken down by shaking. This was accelerated in the presence of ballotini beads but aggregations of bacteria were still present after prolonged agitation. Vigorous shaking, particularly with beads, decreased the viability of the bacteria and optimum viable counts were obtained after manual shaking for half a minute. Immersion in buffered detergent, wash and diluting fluids for up to 2 h promoted release of bacteria from microcolonies but decreased the viability of aerobic and anaerobic pleomorphic rods and a Bacillus strain. There was no significant effect on strains of Micrococcaceae. Prolonged exposure of bacteria from scrub samples to these fluids can thus lead to both quantitative and qualitative alterations in the counts obtained, although these effects may be masked by the continuing release of bacteria from microcolonies. The cup-scrub technique provides a convenient means of quantifying changes in the bovine skin microflora but results obtained from different studies should only be compared if closely similar techniques are used.     

Application of 2H NMR to the study of natural site-specific hydrogen isotope transfer among substrate,medium, and glycerol in glucose fermentation with yeast

2H NMR is a very useful tool in isotope tracing studies. This technique was applied to a quantitative study of a site-specific deuterium affiliation among the substrate, the medium, and a product (glycerol), in glucose fermentation with yeast. The quality of the results depends on the quantitative 2H NMR analysis of glycerol. After comparing several potential analysis probe molecules, the derivative of glycerol, 2,2-dimethyl-1,3-dioxolane-4-methanol, was chosen as the most advantageous. Using this probe in a set of isotope-labeling experiments, we describe how a complete quantitative site-specific hydrogen isotope transfer model, which connects the site-specific isotopic ratios of the substrate, the medium, and the products, can be established. This model can provide information on complex hydrogen transfer mechanisms during biochemical reactions and can be useful for the prediction of site-specific hydrogen isotopic ratios at natural abundance of the products, based on that of the substrate or reactants and the medium.     

THE RETENTION OF 32P-LABELLED RHIZOBIUM BY LEGUME SEED AFTER INOCULATION BY VACUUM TREATMENT

Legume seeds were vacuum treated in a suspension of ³²P-labelled Rhizobium meliloti to find the numbers and uniformity of distribution of bacteria taken up and retained by the seeds after washing. Commercial seed retained 5–12 times as many bacteria as did hand harvested seed. From 5 to 15% of the seeds held 75–90% of the bacteria; vacuum treatment increased uptake by this small group of seeds by 60–300%, but had no significant effect on the majority that acquired relatively few bacteria. Therefore vacuum treatment of seeds during inoculation is likely to affect nodulation on a small proportion of the plants only.     

Plant immunity: a lesson from pathogenic bacterial effector proteins

Phytopathogenic bacteria inject an array of effector proteins into host cells to alter host physiology and assist the infection process. Some of these effectors can also trigger disease resistance as a result of recognition in the plant cell by cytoplasmic immune receptors. In addition to effector-triggered immunity, plants immunity can be triggered upon the detection of Pathogen/Microbe-Associated Molecular Patterns by surface-localized immune receptors. Recent progress indicates that many bacterial effector proteins use a variety of biochemical properties to directly attack key components of PAMP-triggered immunity and effector-triggered immunity, providing new insights into the molecular basis of plant innate immunity. Emerging evidence indicate that the evolution of disease resistance in plants is intimately linked to the mechanism by which bacterial effectors promote parasitism. This review focuses on how these studies have conceptually advanced our understanding of plant–pathogen interactions.