Direct Microscopic Observation Research Articles

Percolation-to-droplets transition during spinodal decomposition in polymer blends, morphology analysis.

Phase separation kinetics of the off-critical mixture of polystyrene and poly(methylphenylsiloxane) is studied by the time-resolved light scattering and optical microscopy. The results from the light scattering experiments are correlated with the images obtained by the optical microscopic observation in order to find characteristic features of the scattering intensity during the percolation-to-droplets morphology transition. At the beginning of the spinodal decomposition process only a bicontinuous network is present in the system and the light scattering intensity has only one peak. The network coarsens and at the same time small droplets appear in the system resulting in a growth of the scattering intensity at very small wave vectors. When the large network starts to break up into disjoint elongated domains a second peak in the scattering intensity appears. Finally, both peaks merge into a single peak at zero wave vector, indicating a complete transformation of elongated domains into spherical droplets of variable sizes. The comparison of the direct microscopic observations with the light scattering spectra shows that the process of breaking up of the bicontinuous network starts when the growth of the first peak, corresponding to the bicontinuous pattern, becomes very slow (essentially pinned down).

Direct Microscopic Observation of Viability of Campylobacter jejuni on Chicken Skin Treated with Selected Chemical Sanitizing Agents

The objective of this research was to determine the effect of chlorine, acidified sodium chlorite, and peracetic acid treatments on viable Campylobacter jejuni located at various depths within follicles or folds of chicken skin. Chicken skin was inoculated with C. jejuni transformed with Pcgfp plasmid (GFP- Campylobacter), which also codes for kanamycin resistance. Effectiveness of sanitizer treatments was determined by plate count. C. jejuni were also observed on chicken skin by confocal scanning laser microscopy, whereby viable and nonviable cells were differentiated by their ability to take up staining with 5-cyano-2,3-ditolyl tetrazolium chloride. Sodium hypochlorite, peracetic acid, and acidified sodium chlorite were each applied at 40 or 100 ppm for 2 or 15 min. Each sanitizer resulted in approximately a 1-log decrease (CFU) when used at 100 ppm for 15 min and no significant decrease when used at 40 ppm for 2 min. Numbers of viable cells observed on the skin by direct microscopic count were similar to numbers obtained by plate count. Although viable counts decreased with sanitizer treatments, the total number of Campylobacter cells (live plus dead) attached to the skin remained unchanged. After each chemical treatment, viable C. jejuni were observed at depths of 0 to 10, 11 to 20, and 21 to 30μm in folds or follicles of chicken skin. Most of the C. jejuni that survived treatment were located at 0 to 10μm depth, which is where most of the viable cells were located before treatment. The inability of chemical sanitizers to effectively eliminate C. jejuni on chicken skin does not appear to be a result of protection by location in feather follicles or other depressions in the skin.

Irreversible adsorption of particles at random-site surfaces.

Irreversible adsorption of negatively charged polystyrene latex particles (averaged diameter 0.9 microm) at heterogeneous surfaces was studied experimentally. The substrate bearing a controlled number of adsorption sites was produced by precovering mica sheets by positively charged polystyrene latex (averaged diameter of 0.45 microm). Positive latex (site) deposition was carried out under diffusion-controlled transport conditions and its coverage was determined by direct particle counting using the optical microscopy. Deposition kinetics of larger latex particles (averaged diameter 0.9 microm) at heterogeneous surfaces produced in this way was studied by direct optical microscope observations in the diffusion cell (under no-convection transport conditions). It was demonstrated that the structure of larger particle monolayers, characterized in terms of the pair correlation function, showed much more short-range ordering than it was predicted for homogeneous surface monolayers at the same coverage. This was found in agreement with theoretical predictions derived from the Monte Carlo simulations. On the other hand, particle adsorption kinetics was quantitatively interpreted in terms of numerical solutions of the governing diffusion equation with the nonlinear boundary condition derived from Monte Carlo simulations. From these kinetic measurements maximum (jamming) coverage of particles was determined in an accurate way by extrapolation. It was concluded that both the monolayer structure and jamming coverage were strongly influenced by the site multiplicity (coordination) effect.

Jamming, two-fluid behavior, and "self-filtration" in concentrated particulate suspensions.

We study the flow of model hard-sphere colloidal suspensions at high volume fraction Phi driven through a constriction by a pressure gradient. Above a particle-size dependent limit Phi(0), direct microscopic observations demonstrate jamming and unjamming-conversion of fluid to solid and vice versa-during flow. We show that such a jamming flow produces a reduction in colloid concentration Phi(x) downstream of the constriction. We propose that this "self-filtration" effect is due to a combination of jamming of the particulate part of the system and continuing flow of the liquid part, i.e., the solvent, through the pores of the jammed solid. Thus we link jamming in colloidal and granular media with a "two-fluid-like" picture of the flow of concentrated suspensions. Results are also discussed in the light of the original experiments of Reynolds on dilation in granular materials.

Fluorescence microscopy for visualization of soil microorganisms?a review

Direct microscopic observation of microorganisms is an important tool in many microbial studies. Such observations have been reported for Protozoa, fungi, inoculated bacteria, and rhizosphere microorganisms but few studies have focused on indigenous bacteria and their spatial relationship within various microhabitats. Principles and applications of epifluorescence microscopy and confocal laser scanning microscopy for visualization of soil microorganisms in situ are reviewed. Both cationic and anionic dyes (also commonly referred to as fluorochromes if they are fluorescent) have been used based on their ability to bind to specific cellular components of microbial cells. Common fluorochromes used for imaging of microbial cells include acridine orange, ethidium bromide, fluorescein isothiocyanate, 5-(4,6-dichlorotriazinyl) aminofluorescein, 4′,6-diamidino-2-phenylindole, europium chelate, magnesium salt of 8-anilino-1-naphthalene sulfonic acid, and calcofluor white M2R. Combining fluorescence staining techniques with soil thin section technology allows one to obtain images of microorganisms in situ. Soil texture and the procedures used for resin embedding are important factors affecting the quality of stained soil thin sections. Indeed, general limitations of applying fluorescence microscopy to soil ecological studies are the non-specific binding of dyes to the soil matrix and the autofluorescence of some soil components. The development of fluorescent in situ hybridization and confocal laser scanning microscopy techniques provides new potential for microbial distribution studies.

Kinetics of particle deposition in the oblique impinging jet cell

A new oblique impinging-jet (OBIJ) cell was developed, suitable for colloid deposition studies at various interfaces. In contrast to previously used orthogonal cells, the OBIJ construction makes possible direct microscope observations of particle deposition on nontransparent substrates. The cell performance was tested by studying kinetics of polystyrene latex particle deposition on mica. Two limiting cell configuration were used in the experiments: (i) the lower position (inverted microscope observation of substrate surface through air) and (ii) the upper position (observation of the substrate surface with adsorbed particles through the suspension layer). The dependence of local mass transfer rate (particle flux) on the position over the substrate surface was studied for various flow Reynolds numbers. It was demonstrated that deposition rate attained maximum at the flow stagnation point whose position was dependent on Re number. Moreover, it was shown that the local flux decreased at much slower rate when moving in the downstream direction, than for previously used impinging-jet cells. Consequently, the area of uniform transport conditions was larger, enabling more precise determination of the limiting particle flux at the stagnation-point. The dependence of the flux on Re number was systematically studied for various ionic strength of the suspension. It was demonstrated, in accordance with previous results for the ordinary impinging-jet, that the flux increased significantly for low ionic strength and high Re number. This phenomenon, referred to as the inverse salt effect, was interpreted in terms of the convective diffusion theory. The governing transport equation originating from this theory was solved numerically, for the region near the stagnation point, using the finite-difference method. These numerical solutions were used for nonlinear fitting of the flow intensity parameter dependence on the Re number. In this way the flow field in the vicinity of the stagnation point was fully characterized. It was concluded that the new cell can be exploited as an effective experimental tool for colloid deposition studies on various substrates.

Particle deposition at electrostatically heterogeneous surfaces

Abstract Irreversible adsorption of negatively charged polystyrene latex particles (averaged diameter 0.9 μm) at heterogeneous surfaces was studied experimentally. The substrate of controlled heterogeneity was produced by covering freshly cleaved mica sheets by positively charged polystyrene latex (averaged diameter of 0.47 μm) to a desired surface coverage. Positive latex deposition was carried out under convection and diffusion-controlled transport conditions and the coverage (defined as heterogeneity degree) was determined by direct particle counting using the optical and electron microscopy. Deposition kinetics of larger latex particles at heterogeneous surfaces produced in this way was studied by using the direct optical microscope observations in the impinging-jet and diffusion cells. It was demonstrated that the initial adsorption rate under the convection-controlled transport attained the limiting value pertinent to homogeneous surfaces for heterogeneity degree of a few per cent. This effect was even more pronounced for diffusion-controlled transport conditions. This behaviour was quantitatively interpreted in terms of the theoretical model considering the coupling between surface and bulk transport of particles. Similarly, the experimental results obtained for higher surface coverage of latex (long adsorption times) were in a good agreement with the generalised random sequential adsorption (RSA) model.

Methods for biofilm analysis on silicone tubing of dialysis machines.

We describe an analytical protocol to study biofilms that develop inside silicone tubing of dialysis machines. This protocol has been set up with the help of a dynamic testing device reproducing dialysis conditions. The methodology includes direct microscopic observation, biofilm removal with an original mechanical biofilm scraper, quantitative analysis with culturable and total bacteria counting, and endotoxin level measurement using the LAL chromogenic kinetic assay. The analytical protocol has been assessed on 13 different clinical tubing samples. Most samples were contaminated by adherent cells and the thickest biofilms were found at the connection between the dialysis water distribution loop and the dialysis machine. The less contaminated samples had been removed from dialysis machines that were decontaminated with citric acid and autoclaving, showing the importance of the decontamination procedure for the prevention of biofilm development. This article shows that easy, rapid, reproducible, and economical methods are applicable for a routine analysis of biofilms that develop on dialysis systems and should be included in the regular control of the microbiological quality of dialysis liquids.

Incidence and diversity of microorganisms within the walls of an active deep-sea sulfide chimney.

A large, intact sulfide chimney, designated Finn, was recovered from the Mothra Vent Field on the Juan de Fuca Ridge in 1998. Finn was venting 302 degrees C fluids on the seafloor and contained complex mineralogical zones surrounding a large open central conduit. Examination of microorganisms within these zones, followed by community analysis with oligonucleotide probes, showed that there were variations in the abundance and diversity of eubacteria and archaea from the exterior to the interior of the chimney. The microbial abundance based upon epifluorescence microscopy and quantitative fatty acid analyses varied from >10(8) cells/g of sulfide 2 to 10 cm within the chimney wall to <10(5) cells/g in interior zones. Direct microscopic observation indicated that microorganisms were attached to mineral surfaces throughout the structure. Whole-cell hybridization results revealed that there was a transition from a mixed community of eubacteria and archaea near the cool exterior of the chimney to primarily archaea near the warm interior. Archaeal diversity was examined in three zones of Finn by cloning and sequencing of the 16S rRNA gene. The majority of sequences from the exterior of the chimney were related to marine group I of the Crenarchaeota and uncultured Euryarchaeota from benthic marine environments. In contrast, clone libraries from interior regions of the chimney contained sequences closely related to methanogens, Thermococcales, and Archaeoglobales, in addition to uncultured crenarchaeal phylotypes obtained from deep subsurface sites. These observations of microbial communities within an active hydrothermal chimney provide insight into the microbial ecology within such structures and may facilitate follow-up exploration into expanding the known upper temperature limits of life.

A quantitative analysis of DNA extraction and purification from compost

We quantified both DNA and humic acid concentrations during the extraction and purification of DNA from compost. The DNA extraction method consisted of bead-beating with SDS for cell lysis, poly(ethylene glycol)-8000 precipitation for preliminary DNA purification, and chromatography on a 10-ml Sephadex G-200 column for final DNA purification. Direct microscopic observation of pre- and post-lysis samples revealed that 95.3±2.3% of native cells was lysed. Sixty-three percent of the original DNA was lost during purification, resulting in a final DNA yield of 18.2±3.8 μg DNA/g of wet compost. The humic acid content was reduced by 97% during the purification steps resulting in a final humic acid concentration of 27±4.7 ng humic acid/μl. The purified DNA fragments were up to 14 kbp in size and were sufficiently free of contaminants to allow both restriction enzyme digestion by four different enzymes and PCR amplification of 16S rDNA.

Order−Disorder Transition in Monolayers of Modified Monodisperse Silica Particles at the Octane−Water Interface

We have studied the effect of particle wettability on the structure of planar monolayers of micron size monodisperse silica particles at the octane−water interface. The particles' wettability was adjusted by silanization of their surfaces with dichlorodimethylsilane; thus their contact angle at the octane−water interface was in the range 70−150°. The modified particles were spread at the interface, and the monolayer structure was studied by direct microscope observation. A very distinct difference in the structure of monolayers depending on the particle contact angle has been observed. Very hydrophobic particles with large contact angles gave well-ordered monolayers at interparticle distances larger than 3 particle diameters. Such well-ordered monolayers in the case of silica particles are reported for the first time. The monolayers of less hydrophobic particles (with contact angles smaller than 115°) were totally disordered and aggregated. The disorder−order transition in the monolayer structure occurred...

Visualization of S100B-positive neurons and glia in the central nervous system of EGFP transgenic mice.

S100B, the EF-hand Ca(++)-binding protein with gliotrophic and neurotrophic properties implicated in the pathogenesis of Alzheimer's disease, is coined as a glial marker, despite its documented presence in rodent brain neurons. We have generated a transgenic mouse whose EGFP reporter, controlled by the -1,669/+3,106 sequence of the murine S100B gene, allows the direct microscopic observation of most S100B-expressing cells in the central nervous system (CNS). From embryonic day 13 onward, EGFP expression was targeted to selected neuroepithelial, glial, and neuronal cells, indicating that cell-specific expression of S100B is regulated at the transcriptional level during development. In adult mice, the highest level of EGFP expression was found in ependymocytes; astrocytes; and spinal, medullar, pontine, and deep cerebellar S100B neurons. Our results, thus, agree with earlier reports suggesting that S100B is not a CNS glial-specific marker. In addition, we detected EGFP and S100B in forebrain neurons previously thought not to express S100B in the mouse, including neurons of primary motor and somatosensory neocortical areas, the ventral pallidum and prerubral field. Another interesting finding was the selected EGFP targeting to neonatal S100B oligodendrocytes and adult NG2 progenitors as opposed to mature S100B oligodendrocytes. This finding suggests that, except for oligodendrocytes at the last stage of myelin maturation, the -1,669/+3,106 sequence of the S100B gene is a useful reagent for driving expression of transgenes in most S100B-expressing cells of mouse brain.

Latex particle adsorption at heterogeneous surfaces

Abstract Irreversible adsorption of colloid particles at heterogeneous surfaces was studied experimentally. The substrate of controlled heterogeneity was produced by covering freshly cleaved mica sheets by colloid iron oxide (hematite) particles to a desired concentration. The particles of averaged diameter of 210 nm were produced by forced hydrolysis of FeCl 3 solutions followed by a hydrothermal ageing. The coverage of colloid hematite particles (heterogeneity degree) was determined by electron and optical microscopy. Adsorption kinetics of polystyrene latex (averaged diameter 0.87 μm) at heterogeneous surfaces was studied by using the direct optical microscope observations in the impinging-jet cell. It was observed that the initial adsorption rate attained the limiting value pertinent to homogeneous surfaces for heterogeneity degree of a few per cent. This behaviour was quantitatively interpreted in terms of the theoretical model considering the coupling between surface and bulk transport of particles. Similarly, the experimental results obtained for higher surface coverage of latex (long adsorption times) were in a good agreement with the random sequential adsorption model.

An experimental approach for direct observation of the interaction of polyanions with sphingosine-containing giant vesicles

A new approach for direct optical microscopy observation of polyanion interactions with bilayers of giant cationic liposomes (GUVs) was suggested. Polyanions as DNA, dextran sulfate (DS), heparin (H) and polyacrylic acids (PA) were locally delivered by a micropipette to a part of a giant unilamellar vesicle membrane. The phenomena were directly observed under optical microscope. GUVs, about 100 μm in diameter, formed of phosphatidylcholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine (Sph), were prepared by electroformation. The effects of water-soluble molecules with high negative linear charge density as dextran sulfate (DS), heparin (H) polyacrylic acids (PA) and adenosine-5′-triphosphoric acid (ATP) were compared with those of DNAs. The resulting membrane topology transformations were monitored in phase contrast, while the DNA distribution was followed in fluorescence. DNA-induced endocytosis-like membrane morphology transformation due to the DNA/lipid membrane local interactions was observed. The DS, H and PA induced membrane topology transformations similar to those of the DNAs, while ATP did not cause any detectable ones. The endocytosis mechanism involves the formation of ordered domains in the GUV membrane where some surface and charge asymmetries between the two membrane monolayers were created. The sizes of created polyanionic/cationic membrane domains depend on the form, length and elasticity of the adsorbed highly charged molecules. Endosome-including capacities of polyanionic molecules depend heavily on the high linear negative charge at a certain length. An original method for direct studying of the DNA/membrane interactions in autoadaptable giant liposome system imitating biological membrane interactions was forwarded. The model observations could also help for understanding events associated with cationic liposome/DNA complex formation in gene transfer processes.

Polystyrene Latex Adsorption at the Gold/Electrolyte Interface

Irreversible deposition of polystyrene latex particles (average diameter, 1.5 μm) on various solid/electrolyte interfaces was studied experimentally by using the direct microscope observation method. The substrate surfaces included bare mica (reference interface), gold covered mica (layer thickness of 50 nm), and solid gold plate. The morphology and thickness of the gold layer on mica was determined by atomic force microscopy. Well-defined transport conditions of particles were created by using the new impinging-jet cell. A characteristic feature of the cell was that the suspension stream was directed obliquely to the interface. This unique characteristic was advantageous allowing one for direct, in situ, observation of particle deposition at metals and other nontransparent interfaces. Experiments performed for various flow intensities indicated that the initial deposition kinetics at all interfaces was identical within the error bounds, in accordance with the model based on the convective–diffusion theory. It was concluded that the limiting flux was governed by the bulk transport rather than by the specific surface interactions.

Low-temperature adiabatic calorimetry of salol and benzophenone and microscopic observation of their crystallization: finding of homogeneous-nucleation-based crystallization

An adiabatic calorimetric and direct microscopic observation of crystallization at low temperatures was performed for salol and benzophenone. Both of the materials in the supercooled-liquid phase exhibited a low-temperature crystallization proceeding in the glass-transition temperature ( Tg) region. The crystallization was observed to proceed as the advance of the crystal front into the liquid phase, and stopped suddenly atT= 227 K and T≈ 215 K for salol and benzophenone, respectively. This anomalous temperature dependence of the crystal-growth rate is a characteristic feature of the homogeneous-nucleation-based (HNB) crystallization, which has been reported for o -terphenyl and triphenylethylene, and the presently observed low-temperature crystallization in salol and benzophenone was concluded to be the HNB crystallization, in which the crystal growth is brought about by the coalescence of crystal nuclei to the crystalline phase on the liquid-crystal interface. The substances exhibiting the HNB crystallization were revealed to have almost the same characteristics with respect to configurational entropy, and undergo the HNB crystallization in the same range of temperature normalized by Tg. These results indicate that the HNB crystallization is potentially a universal phenomenon in fragile liquids, and imply that the process is closely related to the liquid structure changing with temperature.

PREPARATION AND CHARACTERIZATION OF POLYSULFONE AND POLYETHERSULFONE MEMBRANES FOR CALCIUM (Ca2+) AND MAGNESIUM (Mg2+) SEPARATION BY COMPLEXATION ULTRAFILTRATION

ABSTRACT Asymmetric ultrafiltration membranes were synthesized from locally available polysulfone and polyethersulfone polymers using aprotic solvents and organic additives by the phase inversion method. The membranes were characterized in terms of pure water permeability, separation behavior with respect to polyethylene glycols of various molecular weights and electrolytes. The suitability of using polyethyleneimine (PEI) for selective removal of calcium and magnesium salts by an ultrafiltration process was studied in terms of optimum polymer loading at reasonable permeate flux, irreversible adsorptive fouling of the macromolecular ligand on the polymer as functions of solution pH and ionic strength, and metal ion separation as a function of concentration and pressure. Direct electron microscopic observation of fresh, as well as fouled, membranes are presented.

Deposition of Particles in the Impinging-Jet Cell for the High Coverage Regime

Particle deposition in the radial impinging-jet cell for the high coverage regime was studied theoretically and experimentally. A detailed description of the flow distribution in the cell was attained by solving the governing Navier–Stokes equation numerically. The macroscopic flow pattern was decomposed into simpler local flows. It was demonstrated that for tangential distances r/R>0.5 the overall flow at the interface was dominated by the simple shear. The intensity of this flow was calculated numerically as a function of the Reynolds number and the distance from the cell center. Knowing the fluid velocity field the convective diffusion equation was formulated describing a two-dimensional transport of particles. As a result of nonlinearity of the boundary condition this equation was solved in an exact manner for low coverage only. For higher coverage, approximate methods were proposed exploiting the random sequential adsorption (RSA) approach. The validity of the theoretical predictions was verified experimentally using the direct microscope observation method and polystyrene latex particles of the size 0.87 μm. Particle coverage distribution was studied in detail as a function of the Re number governing the local shear rate. It was demonstrated that for low Re number (Re<4) uniform particle monolayers of high coverage can be attained. On the other hand, for Re>8 the particle coverage distribution became nonuniform as a result of the hydrodynamic scattering. This effect, leading to an apparent kinetic saturation of the surface at coverages of a few percents, was quantitatively interpreted in terms of the theoretical model.

Effect of 0.25 T static magnetic field on microcirculation in rabbits.

We showed previously in rabbits that 0.2 and 0.35 T static magnetic field (SMF) modulated systemic hemodynamics by arterial baroreceptors. We now have measured the effect of 0.25 T SMF on microcirculation within cutaneous tissue of the rabbit ear lobe by the rabbit ear chamber (REC) method. Forty experimental runs (20 controls and 20 SMF) were carried out in eight different rabbits with an equal number of control and SMF experiments on each individual. Rabbits were sedated by pentobarbital sodium (5 mg/kg/h, i.v.) during the entire 80 min experiment. SMF was generated by four neodium-iron-boron alloy (Nd2-Fe14-B) magnets (15 x 25 x 30 mm, Neomax, PIP - Tokyo Co., Ltd., Tokyo, Japan), positioned around the REC on the observing stage of an optical microscope. The direct intravital microscopic observation of the rabbit's ear microvascular net, along with simultaneous blood flow measurement by microphotoelectric plethysmography (MPPG), were performed PRE (20 min, baseline), DURING (40 min), and POST (20 min) magnetic field exposure. The control experiments were performed under the same conditions and according to the same time course, but without magnetic field. Data were analyzed comparing MPPG values and percent change from baseline in the same series, and between corresponding sections of control and SMF runs. In contrast to control series (100+/-0.0%-90.0+/-5.4%-87.7+/-7.1%, PRE-EXPOSURE-POST), after magnetic field exposure we observed increased blood flow (100+/-0.0%-117.8+/-9.6%*-113.8+/-14.0%, *P<0.05) which gradually decreased after exposure cessation. We propose that long exposure of a high level nonuniform SMF probably modifies microcirculatory homeostasis through modulation of the local release of endothelial neurohumoral and paracrine factors that act directly on the smooth muscle of the vascular wall, presumably by affecting ion channels or second messenger systems.

Irreversible adsorption of colloid particles at heterogeneous surfaces

Irreversible adsorption of polystyrene latex particles of micrometer size range at heterogeneous surfaces was studied experimentally. Model substrate surfaces of controlled site coverage (heterogeneity degree) used in these studies were produced by preadsorption of positively charged latex particles on mica sheets. Deposition kinetics of latex was studied as a function of the site coverage, particle to site size ratio λ and ionic strength of the colloid suspension. Particle distributions over surfaces and coverage were quantitatively evaluated by the direct microscope observation techniques using the diffusion cell. In this way, pair correlation function for various coverage degree and particle size ratio was evaluated. It also was determined the dependence of the jamming coverage of colloid particles on site coverage and ionic strength of the suspension. It was demonstrated that the decrease in the ionic strength of the suspension resulted in a significant decrease in the jamming coverage. This was attributed to the effect of the electrostatic field generated by the interface whose range was increased for low ionic strength. These experimental data revealed, in accordance with theoretical predictions derived from numerical simulations, that the multiple site coordination exerted a pronounced effect on the jamming coverage and the structure of adsorbed layers. It also was shown that this effect can be regulated by changes in the ionic strength of particle suspensions. This could allow one to produce particle clusters at the surface of targeted composition.