Hydrogen peroxide staining to visualize intracellular bacterial infections of seedling root cells

Visualization of bacteria in living plant cells and tissues is often problematic due to lack of stains that pass through living plant cell membranes and selectively stain bacterial cells. In this article, we report the use of 3,3′‐diaminobenzidine tetrachloride (DAB) to stain hydrogen peroxide associated with bacterial invasion of eukaryotic cells. Tissues were counterstained with aniline blue/lactophenol to stain protein in bacterial cells. Using this staining method to visualize intracellular bacterial (Burkholderia gladioli) colonization of seedling roots of switch grass (Panicum virgatum), we compared bacterial free seedling roots and those inoculated with the bacterium. To further assess application of the technique in multiple species of vascular plants, we examined vascular plants for seedling root colonization by naturally occurring seed‐transmitted bacteria. Colonization by bacteria was only observed to occur within epidermal (including root hairs) and cortical cells of root tissues, suggesting that bacteria may not be penetrating deeply into root tissues. DAB/peroxidase with counter stain aniline blue/lactophenol was effective in penetration of root cells to selectively stain bacteria. Furthermore, this stain combination permitted the visualization of the bacterial lysis process. Before any evidence of H₂O₂ staining, intracellular bacteria were seen to stain blue for protein content with aniline blue/lactophenol. After H₂O₂ staining became evident, bacteria were often swollen, without internal staining by aniline blue/lactophenol; this suggests loss of protein content. This staining method was effective for seedling root tissues; however, it was not effective at staining bacteria in shoot tissues due to poor penetration. Microsc. Res. Tech. 77:566–573, 2014. © 2014 Wiley Periodicals, Inc.     

A technique for the preparation of cross-sectional TEM samples of ZnSe/GaAs heterostructures which eliminates process-induced defects

Cross-sectional transmission electron microscopy (TEM) sample preparation of ZnSe/GaAs epitaxial films is investigated. Conventional argon ion milling is shown to produce a high density (～ 5–8 × 10¹¹/cm²) of small (diameter ～ 60–80 Å) extended defects (stacking faults, microtwins, double positioning twins, etc.). In addition, transmission electron diffraction results indicate a thin ZnO layer can also occasionally form upon ion milling or electron-beam irradiation although the exact conditions for ZnO formation are not well understood. Conventional TEM (amplitude contrast) and high-resolution TEM (phase contrast) imaging in combination with transmission electron diffraction studies were performed to determine the optimum method of removing the ion milling related damage and ZnO layers during sample preparation. HF/HCl, NaOH/H₂O, H₂SO₄/H₂O₂/H₂O and Br₂/CH₃OH etching mixtures as well as low voltage argon or iodine ion milling were studied. A low energy (2 ke V) iodine or argon ion milling step was shown to remove the ZnO layer and reduced the density of the extended defects associated with Ar⁺ ion milling, but was unsuccessful in removing all of the defects. Auger electron spectroscopy results indicate residual iodine was either left on the surface or implanted beneath the surface during iodine ion milling. Etching the XTEM samples in HF/HCl was shown to be effective in removing the ZnO layer but had little or no effect on the ion milling induced defects. Etching the samples in a 0.5% Br₂/CH₃OH solution resulted in complete elimination of the ion milling induced extended defects including the residual defects associated with iodine ion milling. In addition the Br₂/CH₃OH etch produced the best surface morphology. Thus a brief (1–2 seconds) Br₂/CH₃OH etch after conventional preparation (argon ion milling) of cross-sectional ZnSe/GaAs TEM samples appears to be an inexpensive and superior alternative to iodine ion milling.     

Independent trafficking of flavocytochrome b558 and myeloperoxidase to phagosomes during phagocytosis visualised by energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy

When polymorphonuclear leukocytes (PMNs) phagocytose opsonised zymosan particles (OPZ), free radicals and reactive oxygen species (ROS) are formed in the phagosomes. ROS production is mediated by NADPH oxidase (Nox), which transfers electrons in converting oxygen to superoxide (O₂^?). Nox‐generated O₂^? is rapidly converted to other ROS. Free radical‐forming secretory vesicles containing the Nox redox center flavocytochrome b558, a membrane protein, and azurophil granules with packaged myeloperoxidase (MPO) have been described. Presuming the probable fusion of these vesicular and granular organelles with phagosomes, the translation process of the enzymes was investigated using energy‐filtering and energy‐dispersive spectroscopy‐scanning transmission electron microscopy. In this work, the primary method for imaging cerium (Ce) ions demonstrated the localisation of H₂O₂ generated by phagocytosing PMNs. The MPO activity of the same PMNs was continuously monitored using 0.1% 3,3′‐diaminobenzidine‐tetrahydrochloride (DAB) and 0.01% H₂O₂. A detailed view of these vesicular and granular structures was created by overlaying each electron micrograph with pseudocolors: blue for Ce and green for nitrogen (N).     

A single crystal of YALO3: Ce3+ as a fast scintillator in SEM

A new single-crystal scintillator intended for applications in a scanning electron microscope (SEM) is presented. It is a single-crystal disc of yttrium aluminium perovskite activated by trivalent cerium free of traces of Fe ions. The single crystals of YAlO₃: Ce³⁺ (YAP: Ce³⁺) were prepared by the Czochralski method in a reducing atmosphere of Ar and H₂ with an excess amount of Y³⁺ ions. Effective methods of purification and purity control of the raw material are described. The highest achievable concentration of Ce³⁺ ions in as grown crystals amounted to 0.3 wt%, concentration in the melt was 8–9 times higher. The best properties were found with samples of the maximum possible concentration of Ce³⁺. Compared with the previous aluminate Y₃ Al₅ O₁₂: Ce³⁺ (YAG: Ce³⁺) these samples showed higher efficiency, a shorter decay time of luminescence (40 ns) and an emission band in a more advantageous spectral region (378 nm). Because of high resistance to radiation damage, high chemical resistance and applicability to ultra-high vacuum it is also suitable for detection of other kinds of ionizing radiation.     

Histochemical comparison of the nonhost tomato with resistant wheat against Blumeria graminis f. sp. tritici

The nonhost interaction of tomato‐Blumeria graminis f. sp. tritici (Bgt) and resistant host interaction of wheat‐Bgt were compared histochemically. The percentage of appressorium formation had no significant difference on tomato and wheat leaves. Papilla formation occurred earlier and more on host wheat than on nonhost tomato leaves, while the incidence of hypersensitive cell death was much higher in the nonhost interaction. Whole‐cell H₂O₂ accumulation and hypersensitive cell death usually appeared in haustorium‐invaded wheat epidermal cells. In contrast, the vast majority of non‐haustorium epidermal cells were associated with H₂O₂ accumulation and hypersensitive cell death on tomato. Localized H₂O₂ accumulation and hypersensitive response were detected in effective papillae in both interactions. The peak percentage of haustorium formation was less than 7% in the nonhost interaction while reached 43% in the incompatible host interaction. These results indicate that hypersensitive cell death and papillae are likely to play an important role in preventing Bgt penetration and development on tomato and wheat leaves, both defense responses involving H₂O₂ accumulation. This study further implies that the nonhost and incompatible interactions share similar cytological mechanisms. Microsc. Res. Tech. 76:514–522, 2013. © 2013 Wiley Periodicals, Inc.     

Synthesis and characterization of permalloy-reinforced Al2O3 nanocomposite powders by mechanical alloying

Intermetallics of Fe and Ni, which are known as permalloy, are under attention due to their excellent magnetic performance. Besides, mechanical properties of the materials can be improved by decreasing crystallite size of FeNi intermetallics or by reinforcing them with hard secondary phases such as Al₂O₃. In this study, FeNi–Al₂O₃ nanocomposite powders with three different compositions were successfully synthesized through mechanical alloying of Fe₂O₃, Ni, and Al powders mixture. Characterization of the samples was accomplished by scanning electron microscopy, transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy and X-ray diffraction. Effects of various parameters such as chemical composition of received materials, milling time, and annealing on the phase evolution, morphology, and microhardness of samples were investigated. It was found that by the addition of Fe as diluent, the required milling time for formation of FeNi intermetallic increased. By increasing milling time, mean crystallite size of FeNi decreased and reach to about 28 nm for FeNi-30 wt% Al₂O₃ nanocomposite powder sample. TEM observations also showed that in situ-formed Al₂O₃ particles, with particle size of about 65 nm, were uniformly dispersed within FeNi matrix.     

A method for abolishing the nonspecific binding artifacts produced by the peroxidase-antiperoxidase complex (PAP) in immunostained mucous epithelia

The presence of nonspecific staining artifacts is a potential problem in ultrastructural immunocytochemistry. In the course of staining for lysozyme in the human and rabbit endocervix, the peroxidase-antiperoxidase complex (PAP) was found to bind to mucous granules in a selective and nonspecific manner. Sections etched in 10% aqueous H₂O₂, incubated for 5 minutes in 1:50 PAP in Tris-buffered saline (TBS) and subsequently treated with 3, 3′-diaminobenzidine-H₂O₂, revealed a staining precipitate within the matrix of mucous granules. The same selective and nonspecific staining could also be visualized when horseradish peroxidase (1 mg/ml) was substituted for PAP in the immunocytochemical sequence. Thus, this staining method made the reliable demonstration of the desired antigen difficult. The affinity of PAP for mucous granules can be completely eliminated by subjecting sections, previously etched in H₂O₂ to expose the antigenic sites, to a 3-minute incubation in immunoglobulin (1:10 human IgA, 1:5 human IgG, or 1:5 antihuman IgG) immediately prior to using PAP in the standard immunocytochemical staining sequence. The use of this modification to Sternberger's unlabeled antibody-enzyme method is recommended because it allows for the elimination of all nonspecific staining artifacts without interfering with specific localization by the primary antibody. The mechanism that causes nonspecific binding of peroxidase to mucous granule constituents is unclear, although carbohydrate binding may play a role in the interaction between mucous granules and peroxidase.     

Vacuum Methods in Electron Microscopy. By Wilbur C. Bigelow

Microstructural evolution in two in situ reinforced composite systems, one produced by partial reduction and the other by grain growth, has been examined over a range of size scales using light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Metal-ceramic matrix composites were formed by the partial reduction of Al₂O₃–Cr₂O₃ solid solutions to form Cr metal particles in an Al₂O₃–Cr₂O₃ matrix which had a lower Cr₂O₃ concentration. In a second system, grain growth of Si₃N₄ during liquid phase sintering produced large, whisker-like grains in a fine-grained Si₃N₄ matrix, bonded by an oxynitride phase. The mechanisms controlling microstructural evolution in these two systems were examined.     

Determination of tetracyclines by novel singlet-oxygen mediated cerium(IV) chemiluminescence

Novel chemiluminescence mechanisms of cerium(IV) and five tetracyclines in the absence and presence of Tween 80 are reported. The role of tetracyclines in the present chemiluminescence system was studied in detail by ultraviolet spectra and the singlet oxygen ¹O₂ probe 2-methyl-6-(4-methoxyphenyl)-3, 7-dihydroimidazo[1,2-a]-pyrazin-3-one hydrochloride. It is the first report that cerium(IV) reacted with tetracyclines producing singlet oxygen ¹O₂ after breaking the organic skeleton and decomposition of peroxyacetic acid in the absence of the surfactant Tween. Furthermore, by examining the effects of sodium azide and the chemiluminescence spectra, the results indicated that the maximum chemiluminescence emission at approximately 480?nm was due to dimer emission of singlet oxygen ¹O₂ from the reaction between cerium(IV) and tetracyclines as well as the reaction of cerium(IV) and Tween 80, respectively. Moreover, the chemiluminescence signals were unaffected when the reaction was performed under anaerobic condition, indicating that dissolved oxygen was independent of this system. Under the optimal conditions, the analytical characteristics and parameters of the cerium(IV)-Tween 80-tetracyclines chemiluminescence system were investigated. The present method was successfully employed for the determination of tetracyclines in milk.     

Imaging of reactive oxygen species burst from mitochondria using laser scanning confocal microscopy

Objective: Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). Methods: Cardiac H9c2 cells were exposed to H₂O₂ (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H₂DCFDA). Results: A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H₂O₂ compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H₂DCFDA. Conclusions: Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to. Microsc. Res. Tech. 76:612–617, 2013. © 2013 Wiley Periodicals, Inc.     

Insight of EDX analysis and EFTEM: Are spherocrystals located in strombidae digestive gland implied in detoxification of trace metals?

Digestive tubules of Strombidae are composed by three cell types: digestive cells, vacuolated cells, and crypt cells. The last one is characterized by the presence of intracellular granules identified as spherocrystals. Such structures are known to occur in basophilic cells of gastropod digestive gland, where they are supposed to be involved in the regulation of some minerals and in detoxification. In this study, energy‐dispersive X‐ray analysis (EDX) and energy filtered transmission electron microscopy (EFTEM) were used to determine the elemental content of spherocrystals in two Strombidae, Strombus gigas and Strombus pugilis. In freshly collected individuals of both species, the following elements were detected: Ca, Fe, Mg, P, and Zn. Aluminum and Mn were also detected in S. gigas. Their presence in spherocrystals indicates that, in Strombidae, spherocrystals are involved in the regulation of minerals and essential trace metals. In order to answer the question “are spherocrystals involved in nonessential trace metals scavenging?,” artificial cadmium and lead exposure by both waterborne and dietary pathways was applied to S. pugilis. No evidence of cadmium (Cd(NO₃)₂) or lead (Pb(NO₃)₂) provided by food was found in spherocrystals. Cadmium provided in water (Cd(NO₃)₂ and CdCl₂) causes structural modifications of the digestive gland; however, this element was not trapped in spherocrystals. These results suggest that spherocrystals are not involved in detoxification of such nonessential trace metals. Microsc. Res. Tech., 2011. © 2011 Wiley‐Liss, Inc.     

How does antiseptic mouthwashes against SARS-COV-2 affect the bond strength of universal adhesive to enamel?

This study compares the effect of different mouthwashes that have been recommended during the Coronavirus disease 2019 (COVID-19) pandemic on shear bond strength (SBS) of universal adhesive to enamel in regards to self-etch (SE) and etch-and-rinse (ER) modes. Flat enamel surfaces were obtained from 100 sound human maxillary central incisors. They were randomly allocated to five groups according to the different mouthwashes (no mouthwash/control [Ctrl], 0.2% chlorhexidine 1.5% hydrogen peroxide [H₂O₂], 0.2% povidone-iodine [PVP-I], Listerine [L]), and adhesive application modes (ER and SE) (n = 10). After the application of a universal adhesive (single bond universal), composite resin (Filtek Z250) was bonded by a cylinder-shaped mold (height: 2 mm, diameter: 2.4 mm). They were subjected to SBS test using a universal testing machine (AGS-X, Shimadzu Corp.) (crosshead speed: 1 mm/min). The resin–enamel interfaces were observed with a scanning electron microscope (SEM). The semiquantitative chemical microanalyses were performed with energy-dispersive spectroscopy (EDS). The data were statistically analyzed by two-way analysis of variance and Bonferroni test (p < .05). In SE mode, Group Ctrl revealed significantly higher SBS than all mouthwash groups (p < .05). In ER mode, Group Ctrl showed significantly higher SBS than H₂O₂ and PVP-I groups (p < .05). ER mode caused significantly higher SBS than SE mode in all mouthwash groups (p < .05). The SEM observations highlighted that Group Ctrl had a regular and intact hybrid layer with resin tag formation while the H₂O₂ and PVP-I groups exhibited a thin hybrid layer in both modes. EDS analysis indicated that in SE mode, all mouthwash groups presented increased O content compared to Group Ctrl. H₂O₂ and PVP-I that were suggested for preprocedural use during the COVID-19 pandemic, reduced the enamel bond strength of the universal adhesive in ER mode.     

The influence of alumina on mechanical and tribological characteristics of graphite particle reinforced hybrid Al-MMC

We consider the influence of alumina (Al₂O₃) particles on mechanical and tribological properties of aluminum hybrid metal matrix composites (MMC). Various weight fraction of Al₂O₃ (5, 10 and 15%) and constant weight fraction of graphite (5%) were used to fabricate composites by stir casting method. The effect of Al₂O₃ content on hardness, density and specific wear rate is evaluated. A wear test was performed using central composite design matrix on a pin-on disc apparatus at room temperature for constant sliding distance of 1000 m. The sliding speed, load and weight fraction of Al₂O₃ were the process variables. The results show that the hardness and density increase with increase in Al₂O₃ content. From the analysis of variance (ANOVA), load is the dominant factor that affects the specific wear rate of hybrid composites followed by speed and weight fraction of Al₂O₃. Based on desirability approach, the improvement in the wear resistance of the composites became more prominent at high speed, high load and high weight fraction of Al₂O₃. The worn surface of the pin was examined using scanning electron microscope (SEM) which indicates that the wear mechanism of composites is mostly abrasive wear followed by oxide wear.     

Study on tribological properties of Al2O3 ceramics/1Cr18Ni9Ti stainless steel rubbing pairs in H2O2 solutions

The tribological properties of (Al₂O₃) ceramics/1Cr18Ni9Ti stainless steel rubbing pairs were investigated using a MMW‐1 tribo‐tester under pure water and different concentrations of hydrogen peroxide (H₂O₂) solutions. A comparison analysis of the friction coefficient, wear mass loss and worn surface topographies was conducted. The wear mass loss of the 1Cr18Ni9Ti stainless steel ring sample in all H₂O₂ solutions was greater than that in pure water. The wear mass loss was the highest in 70% H₂O₂ solution. The comprehensive analysis showed that the main wear mechanisms between the Al₂O₃ ceramics/1Cr18Ni9Ti stainless steel rubbing pairs were severe adhesive, abrasive and ploughing wear in the H₂O₂ solutions. It is believed that the knowledge gained in this study will provide the theoretical data and guide for the optimisation of the rubbing pairs in the H₂O₂ solutions to be used for the lubrication of some moving parts in the propulsion system of the launch vehicle. Copyright © 2010 John Wiley & Sons, Ltd.     

Forming limit diagram of aluminum/copper bi-layered tubes by bulge test

Chemo-mechanical-grinding (CMG) is a hybrid process which integrates chemical reaction and mechanical grinding between abrasives and workpiece into one process. It has been successfully applied into manufacturing process of silicon wafers where both geometric accuracy and surface quality are required. This paper aims to study the potential of CMG process in manufacturing process of single crystal sapphire wafers. The basic material removal mechanism in terms of chemical effect and mechanical effect in CMG process has been analysed based on experiment results of two different kinds of CMG wheels. The experiment results suggest that chromium oxide (Cr₂O₃) performs better than silica (SiO₂) in both material removal rate (MRR) and surface quality. It also reveals that, no matter under dry condition or wet condition, CMG is with potential to achieve excellent surface quality and impressive geometric accuracy of sapphire wafer. Meanwhile, test result by Raman spectrum shows that, by using Cr₂O₃ as abrasive, the sub-surface damage of sapphire wafer is hardly to be detected. Transmission electron microscopy (TEM) tells that the sub-surface damage, about less than 50 nm, might remain on the top surface if chemical effect is not sufficient enough to meet the balance with mechanical effect in CMG process.     

Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

The application of high resolution electron microscopy to the study of oxidation

High resolution electron microscopy has been used to examine both the similarities and differences in the growth morphologies of Fe₂O₃, Cr₂O₃ as formed on iron alloys, and MgO as formed by burning Mg in moist air. A general discussion is also given of the relative advantages and disadvantages of a variety of TEM techniques as applicable to the study of oxidation phenomena.     

Experimental and Calculational Studies of the Interactions of BF3 with Fluoroethers

BF₃ was co-condensed with (C₂H₅)₂O, (CF₃CH₂)₂O, and (C₂F₅)₂O in excess argon at 15 K. Infrared spectra of the matrices showed a definite Lewis acid-base interaction between BF₃ and diethyl ether; a weak but definite interaction with bis(2,2,2,-trifluorodiethyl)ether, and no observable interaction with perfluorodiethyl ether. Molecular orbital (MO) calculations complemented the experimental observations by revealing that fluorine atoms on the ethers decreased electron localization about the oxygen atom. Thus, the experimental data and MO calculations indicated a clear trend between strength of interaction with BF₃ and the degree of ether F substitution. The implications of the results for commercial perfluoro ether lubricant/metal oxide surface interactions are discussed.     

Effects of prolonged use of over-the-counter bleaching agents on enamel: An in vitro study

This study evaluated the effects of four over-the-counter (OTC) bleaching products on the properties of enamel. Extracted human molars were randomly assigned into four groups (n = 5): PD: Poladay (SDI), WG: White Teeth Global (White Teeth Global), CW: Crest3DWhite (Procter & Gamble), and HS: HiSmile (HiSmile). The hydrogen peroxide (H₂O₂) content in each product was analyzed via titration. Twenty teeth were sectioned into quarters, embedded in epoxy resin, and polished. Each quarter-tooth surface was treated with one of the four beaching times: T0: control/no-bleaching, T14: 14 days, T28: 28 days, and T56: 56 days. Materials were applied to enamel surfaces as recommended. Enamel surfaces were examined for ultramicrohardness (UMH), elastic modulus (EM), superficial roughness (Sa), and scanning electron microscopy (SEM). Ten additional teeth were used to evaluate color and degree of demineralization (DD) (n = 5). Data were statistically tested by two-way ANOVA and Tukey's tests (α = 5%). Enamel surfaces treated with PD and WG presented UMH values significantly lower than the controls (p < .05). Elastic modulus (E) was significantly reduced at T14 and T28 for PD, and at T14 for HS (p < .05). A significant increase in Sa was observed for CW at T14 (p < .05). Color changes were observed in the PD and WG groups. Additionally, DD analysis showed significant demineralization at T56 for CW. Overall, more evident morphological alterations were observed for bleaching products with higher concentrations of H₂O₂ (p < .05), PD, and WG. Over-the-counter bleaching products containing H₂O₂ can significantly alter enamel properties, especially when application time is extended.