ULTRATHIN FROZEN SECTIONS : I. Methods and Ultrastructural Preservation

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70°C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35°C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.     

A Blaze-Dry Spreading Procedure for the Electron Microscopy of Chromosomes from Acid Alcohol-Fixed Human Lymphocytes

A procedure is described for the blaze-drying of human lymphocyte chromosomes on carbonized Parlodion film. Films are prepared by applying Parlodion solution to sheets of freshly cleaved mica. Damage to the film during blaze-drying is prevented by chilling the mica sheets on dry ice before flaming. After spreading, the film and metaphases are floated free from the mica and transferred to a slide of Formvar-coated electron microscope grids. The resulting preparations yield complete metaphase spreads and banded chromosomes morphologically similar to those observed with the light microscope.     

Fabrication of carbon films with ∼500 nm holes for cryo-EM with a direct detector device

Single particle electron cryomicroscopy (cryo-EM) is often performed using EM grids coated with a perforated or holey layer of amorphous carbon. Regular arrays of holes enable efficient cryo-EM data collection and several methods for the production of micropatterned holey-carbon film coated grids have been described. However, a new generation of direct detector device (DDD) electron microscope cameras can benefit from hole diameters that are smaller than currently available. Here we extend a previously proposed method involving soft lithography with a poly(dimethylsiloxane) (PDMS) stamp for the production of holey-carbon film coated EM grids. By incorporating electron-beam (e-beam) lithography and modifying the procedure, we are able to produce low-cost high-quality holey-carbon film coated EM grids with ∼500 nm holes spaced 4 μm apart centre-to-centre. We demonstrate that these grids can be used for cryo-EM. Furthermore, we show that by applying image shifts to obtain movies of the carbon regions beside the holes after imaging the holes, the contrast transfer function (CTF) parameters needed for calculation of high-resolution cryo-EM maps with a DDD can be obtained efficiently.     

Preparation of macromolecular complexes for cryo-electron microscopy

This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.     

Glucose sensor using a microfabricated electrode and electropolymerized bilayer films

A new type miniaturized glucose sensor with good selectivity and stable current response has been developed. The structure consists of a recessed rectangular microfabricated platinum electrode, inner layer of two electropolymerized nonconducting films, and outer bilayer of poly(tetrafluoroethylene) (Teflon) and polyurethane (PU) films. Glucose oxidase (GOx) is entrapped during the electropolymerization of a poly(m-phenylenediamine) (PMPD) film in an acetate buffer (AB) solution, on which a highly interference-resistive PMPD film is deposited in a phosphate buffered saline (PBS) solution. The second PMPD film causes no significant decrease in accessibility of glucose to GOx. The inner layer maintains less than 1% permeability to acetaminophen for 12 days. The fairly adhesive outer layer allows stable current response. Due to high permeability, the information about enzyme activity can be obtained without serious error in spite of outer layer intervening between enzymes and solution. The apparent Michaelis-Menten constant and the maximum steady-state current density were 24 mM and 80 microA cm(-2), respectively.     

Accurate Placement of Ultrathin Sections on Grids; Control by Sol-Gel Phases of a Gelatin Flotation Fluid

A 3% solution of gelatin in a petri dish at 25-30 C. provides a liquid, viscous surface upon which ultrathin sections can be floated. On cooling, the gelled substratum immobilizes the sections, allowing grids to be placed on them with any desired grid-to-section orientation. When the gelatin is remelted, the sections remain attached to the grids. After draining, traces of gelatin adhering to the grids are removed by flotation (section side down) for 30 min on 2% acetic acid at 60 C. This is followed by flotation for 3-5 min on Tris buffer, pH 7.1, and then on distilled water for 30 min—both treatments at 60 C. The technique is particularly useful for mounting serial sections.     

Optimization of airborne endotoxin exposure assessment: effects of filter type, transport conditions, extraction solutions, and storage of samples and extracts

Endotoxin exposure occurs in homes and occupational environments and is known to cause adverse health effects. In order to compare results from different studies and establish standards, airborne endotoxin exposures should be assessed using standardized methods. Although the European Committee for Standardization (CEN) developed guidelines for endotoxin exposure assessment, these leave room for individual interpretation. The influence of methods of sampling, extraction, and analysis has never been investigated in a full experimental design. Thus, we sought to fully elucidate the importance of all facets of endotoxin assessment. Inhalable dust samples collected simultaneously were used to investigate the effects on and interactions with airborne endotoxin concentration in two working environments of filter type (glass fiber or Teflon), transport conditions (with/without desiccant), sample storage (-20 or 4 degrees C), extraction solution (pyrogen-free water [PFW] or PFW plus 0.05% Tween 20), extract storage (-20 or 4 degrees C), and assay solution (PFW or PFW plus 0.05% Tween 20). Four hundred samples were collected and randomly distributed over the 20 combinations of treatments. There were no differences found for transport conditions and storage temperature of extracts. Also, no interactions between study variables existed. Sampling on glass-fiber filters, storage of samples in the freezer, and extraction in PFW plus 0.05% Tween 20 resulted in 1.3-, 1.1-, and 2.1-fold-higher estimated endotoxin concentrations, respectively. Use of PFW plus 0.05% Tween 20 in the assay solution had an additive effect. Thus, this study investigated gaps in the CEN protocol and provides data with which to fully specify a protocol for standardization of endotoxin exposure assessment.     

Evaluation of sorting grids for deepwater rose shrimp (Parapenaeus longirostris) in the Eastern Mediterranean demersal trawl fishery

The study aimed to evaluate sorting grids of 10 and 15 mm bar spacing specifically for separation of deep water rose shrimp, but including other species, in a Mediterranean multispecies demersal trawl fishery. Data were collected 15–25 October 2008 in S??ac?k Bay, Eastern Aegean Sea with the commercial trawler, ‘Hapulo?lu’, using a modified bottom trawl net. A total of 22 valid hauls (12 with 10 mm, ten with 15 mm grids) were obtained. The separation rate of anglerfish was highest, with 100% for weight and number in both codends among all species. Grid elimination of broadtail short‐fin squid showed differences between 95.3 and 80.3% in terms of weight, and 89.7 and 66.2% in terms of number for 10 and 15 mm, respectively. Separation ratios for hake, silver scabbard fish, and horse mackerel were between 96.3 and 100% in terms of weight, and 92.2 and 100% in terms of number in both codends. Shrimp separation was in total calculated as 60.8 and 37.0% by number, and 70.7 and 44.4% by weight in 10 and 15 mm bar spacing trawl grids, respectively, demonstrating that substantial improvement in species selectivity (deep water rose shrimp from others) is possible to achieve by adding sorting grids in the Mediterranean demersal trawl fishery. To optimize the overall selection performance in such fisheries, a variety of grid systems, bar spacings and materials as well as the economic consequences need to be evaluated.     

Microthrix parvicella, a filamentous bacterium from activated sludge: growth on Tween 80 as carbon and energy source

Microthrix parvicella, cultivated in a medium with Tween 80 and Casamino acids, utilized only the oleic acid moiety of Tween 80 as carbon and energy source. The cell yield from Tween 80 was about 0.32 g dry weight of cells per g of Tween 80 consumed. As only the oleic acid moiety of Tween 80 was utilized, the cell yield from oleic acid was 1.3 g dry weight of cells per g oleic acid consumed. The amount of carbon produced as CO2 was less than 30% of the oleic acid-carbon and this low value was in agreement with the high cell yield. In batch culture M. parvicella stored large amounts of lipid material during the early growth phase. The fatty acids of the lipid globules were similar to the fatty acids supplied as carbon source. The percentage composition of the biomass changed to give C/N percentage ratios of about 15 during the early growth phase due to the high concentration of internal lipids and the low concentration of protein. The growth rate in batch culture was about 0.016 h-1 but was affected by the concentration of Casamino acids in the medium.     

I. Novel capacitative electrode with a wide frequency range for measurements of flash-induced changes of interface potential at the oil-water interface Mechanical construction and electrical characteristics of the electrode

The mechanical construction and the electrical properties of a new type of capacitative electrode for the oil-water interface are described. The electrode is designed to detect changes of the interface potential induced by photochemical, photophysical, and photobiological reactions occurring at the interface. The construction is based on capacitative coupling of two aqueous compartments separated by a thin Teflon film. Thereby, the oil-water interface is in horizontal position and the electrode is placed with its planar bottom about 10 μm above the interface. A main feature of the electrode is the transparency to visible light which is achieved by having a clear electrolyte solution in the inner compartment of the capacitative electrode.The aqueous subphase and the inner electrolyte are connected with AglAgCl electrodes to voltage amplifiers. The capacitative electrode is best operated under open circuit conditions. The frequency range experimentally verified is 500 $\text{MHz}\text{\$}\text{?}\text{?}{}_{\mathrm{<mtext>3</mtext><mtext>dB</mtext>}}\text{\$}\text{?}\text{0.03}\text{Hz}$. The sensitivity is mainly determined by the noise of the electronic amplifiers, typical 50–100 μV.     

Photoresist-Free Patterning by Mechanical Abrasion of Water-Soluble Lift-Off Resists and Bare Substrates: Toward Green Fabrication of Transparent Electrodes

This paper describes the fabrication of transparent electrodes based on grids of copper microwires using a non-photolithographic process. The process—“abrasion lithography”—takes two forms. In the first implementation (Method I), a water-soluble commodity polymer film is abraded with a sharp tool, coated with a conductive film, and developed by immersion in water. Water dissolves the polymer film and lifts off the conductive film in the unabraded areas. In the second implementation (Method II), the substrate is abraded directly by scratching with a sharp tool (i.e., no polymer film necessary). The abraded regions of the substrate are recessed and roughened. Following deposition of a conductive film, the lower profile and roughened topography in the abraded regions prevents mechanical exfoliation of the conductive film using adhesive tape, and thus the conductive film remains only where the substrate is scratched. As an application, conductive grids exhibit average sheet resistances of 17 Ω sq^–1 and transparencies of 86% are fabricated and used as the anode in organic photovoltaic cells in concert with the conductive polymer, poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). Compared to devices in which PEDOT:PSS alone serves as an anode, devices comprising grids of copper/nickel microwires and PEDOT:PSS exhibit lowered series resistance, which manifests in greater fill factor and power conversion efficiency. This simple method of forming micropatterns could find use in applications where cost and environmental impact should be minimized, especially as a potential replacement for the transparent electrode indium tin oxide (ITO) in thin-film electronics over large areas (i.e., solar cells) or as a method of rapid prototyping for laboratory-scale devices.     

On Auxin-Induced pH Drop and on the Improbability of its Involvement in the Primary Mechanism of Auxin-Induced Growth Promotion

When sunflower hypocotyl segments were floated on a solution containing potassium, auxin brought about a pH drop in the medium. The effect was detectable about 2–3 hours after the application of the auxin, and persisted during the 26 hours of the experiment. When ammonium was applied instead of potassium, an auxin-induced pH drop was also noted in the first hours of the incubation. Afterwards, the pH of the auxin-treated medium became higher than that of the control solution. It is suggested that the difference in the action of the auxin in the presence of the two cations is related to the contrast between its effects on their absorption. The data are discussed in relation to the recently proposed hypothesis that auxin-induced growth promotion is a result of an auxin-induced pH drop in a cell wall compartment. It is concluded that the results do not support the hypothesis.     

Use of protein A in the serum-in-agar diffusion method in immune electron microscopy for detection of virus particles in cell culture

A modified technique using protein A in the serum-in-agar (SIA) method for immune electron microscopy (IEM) was presented. Grids coated with staphylococcal protein A were floated on samples mounted on agar containing 2% antiserum and incubated at 37 C, for 60 min. After washing and staining, the grids were observed in an electron microscope. The effects of protein A on virus detection were evaluated using poliovirus and bovine rotavirus infected cell culture fluids. The results showed that the technique using protein A (PA-SIA) had at least 10-fold higher sensitivity for virus detection than the original SIA. The optimal concentration of protein A was 1 to 10 micrograms/ml for coating the grids to trap virus particles. The PA-SIA method was also compared with immunosorbent electron microscopy (ISEM). The former showed higher or at least the same sensitivity and some advantages in detecting antigen-antibody reaction than the latter method. These results indicate that our PA-SIA method may be superior to other IEM techniques presented previously for the detection and identification of viruses.     

In Situ Multiple Sampling of Attached Bacteria for Scanning and Transmission Electron Microscopy

An in situ electron microscope sampling technique for characterizing cells attached to smooth surfaces is demonstrated with an ultraviolet-induced mutant of Streptococcus mutans. The sterilized sampling unit consists of a 9 cm plastic Petri dish containing a glass slide, a 12 mm round coverglass, and a coverglass with Formvar-carbon coated copper grids. After the bacterial culture in a liquid medium is incubated in the Petri dish, the slide with attached bacteria is washed in double-distilled water, air-dried, coated with platinum and carbon, and processed for replicas and shadowed specimens for transmission electron microscopy. The coverglass is similarly washed, fixed in 2% glutaraldehyde, air- or freeze-dried, coated with palladium/gold, and examined in the scanning electron microscope. The coverglass with grids is rinsed in double distilled water, the grids are transferred to a filter paper and stained with a loopful of 2% phosphotungstic acid at pH 5.5. The bacteria growing on the surface of the plastic Petri dish are fixed, dehydrated, and embedded in situ with Epon. Sectioned and stained specimens are then examined in the transmission electron microscope. This procedure also appears useful with such other attached systems as normal or infected tissue culture cells.     

表面活性剂对出芽短梗霉多糖生产影响的研究

研究了表面活性剂对出芽短梗霉细胞培养过程中多糖释放的影响。在摇瓶中,比较添加0.05%(w/v)的Tween 80、Tween 60、Tween 40,结果显示几种表面活性剂均能促进细胞释放多糖,其中以Tween 80的效果最佳。在5L发酵罐中,以100g/L玉米粉水解液做碳源的出芽短梗霉细胞培养液中分别添加了表面活性剂Tween 80 0.01%、0.05%、0.1%,其中以添加Tween 800.05%时的效果最好,与不添加表面活性剂相比多糖产量提高25%左右,发酵周期缩短了将近2d。     

Novel Strategy to Fabricate Floating Drug Delivery System Based on Sublimation Technique

The present study aims to develop floating drug delivery system by sublimation of ammonium carbonate (AMC). The core tablets contain a model drug, hydrochlorothiazide, and various levels (i.e., 0–50% w/w) of AMC. The tablets were then coated with different amounts of the polyacrylate polymers (i.e., Eudragit® RL100, Eudragit® RS100, and the mixture of Eudragit® RL100 and Eudragit® RS100 at 1:1 ratio). The coated tablets were kept at ambient temperature (25°C) or cured at 70°C for 12 h before further investigation. The floating and drug release behaviors of the tablets were performed in simulated gastric fluid USP without pepsin at 37°C. The results showed that high amount of AMC induced the floating of the tablets. The coated tablets containing 40 and 50% AMC floated longer than 8 h with a time-to-float of about 3 min. The sublimation of AMC from the core tablets decreased the density of system, causing floating of the tablets. The tablets coated with Eudragit® RL100 floated at a faster rate than those of Eudragit® RS100. Even the coating level of polymer did not influence the time-to-float and floating time of coated tablets containing the same amount of AMC, the drug release from the tablets coated with higher coating level of polymer showed slower drug release. The results suggested that the sublimation technique using AMC is promising for the development of floating drug delivery system.     

Wire-Loop Application of Liquid Emulsion to Slides for Autoradiography in Light Microscopy

This is an adaptation of procedures used to prepare autoradiographs for electron microscopy to light microscopy. A rectangular wire loop slightly larger than the slide is used. The liquid emulsion is prepared by mixing 1 volume of a 0.1% solution of Dreft or Ivory Snow with 2 volumes of Kodak NTB3 nuclear track emulsion. The slide to be coated is placed on a cork stopper supported by a glass plate 61 cm from the safelight. The loop was withdrawn from the liquid emulsion, with the plane of the loop parallel to the surface of the liquid, placed directly over the slide and slowly lowered so that the film (in the sol state) was broken by the slide. If desired, the film can be allowed to gel before application to the slide by waiting approximately 40 sec after the loop is removed from the liquid emulsion. That the developed emulsion is consistently uniform was indicated by a thickness of 0.4 ± 0.02 μm. Diluted emulsion can be stored after use for 1-2 wk at 2-8 C and used a second time before discarding. The identification of human D group chromosomes labeled with tritiated thymidine as well as prescreening patients for the Lesch-Nyhan syndrome, which utilizes tritiated hypoxanthine labeling, have been successfully carried out by applying the emulsion in the sol state.     

Extraction of penicillin-G by aqueous two-phase partition

Summary Pure potassium penicillin-G solution and penicillin-G fermentation broth were extracted using the aqueous two-phase systems of poly-ethylene glycol and salts. The partition coefficients of penicillin-G were above 10 in both pure solution and broth systems. The partition coefficients of phenyl acetic acid were about 0.25. Cell mass and solid residue in the broth system were partly concentrated around the interfacial region and partly settled to the bottom when under 1000 × g centrifugal force. Accordingly, this technique is a promising alternative for the recovery of penicillins.     

Selective medium for the isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum

A medium for the selective isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) was developed, based upon the ability of these mycobacteria to utilize Tween 80 as sole carbon source and grow optimally at pH 5.5 on a simple mineral salts medium. Representative MAIS strains had higher efficiencies of plating on the Tween 80 medium compared with Middlebrook 7H10. It was shown that nonmycobacterial organisms in natural waters had lower efficiencies of plating on the Tween 80 medium and smaller colonies, thus allowing direct isolation and enumeration of the slowly growing mycobacteria without overgrowth.     

A new technique for electron microscopic dry-mounting radioautography of soluble-compounds

Summary A new technique for dry-mounting electron microscopic radioautography of water soluble-compounds has been established.Tissues containing labelled compounds are cut as small as less than 1 mm in diameter, plunged into isopentane cooled to about –160° C with liquid nitrogen, and frozen-dried at –50° C for 24–48 hours. After drying, the tissues are embedded in Epon, which is polymerized according to the conventional procedure.Ultrathin sectioning is accomplished with ethylene glycol instead of water in the knife trough so that water is not involved in the floatation and expansion of the ultrathin sections. Sections are picked up on collodion coated grid meshes from ethylene glycol.Radioautographic emulsion is diluted 1 part in 10 parts with distilled water at 40° C. Ten ml of the diluted emulsion is added with 0.2 ml of 2 per cent aqueous solution of dioctyl sodium sulfosuccinate. A thin film of the emulsion is obtained by dipping a platinum wire loop into the emulsion. The loop is set on a flat surface of a desk for air-drying. The dried film is then applied to the mesh and it is warmed at 37° C for 1 hour in order to help the film to adhere to the mesh. Thus, dry-mounting of packed monolayers of radioautographic silver crystals can be constantly achieved. The mesh is then exposed, developed and stained according to the conventional technique.