Use of a modified Robbins device to directly compare the adhesion of Staphylococcus epidermidis RP62A to surfaces

Staphylococcus epidermidis is a frequent cause of infection associated with the use of biomedical devices. Flow cell studies of the interaction between bacteria and surfaces do not generally allow direct comparison of different materials using the same bacterial suspension. The use of a modified Robbins Device (MRD) to compare the adhesion to different surfaces of Staph. epidermidis RP62A grown in continuous culture was investigated. Adhesion to glass was compared with siliconized glass, plasma-conditioned glass, titanium, stainless steel and Teflon. Attachment to siliconized glass was also compared with glass under differing ionic strength, and divalent cation concentrations. Both the differences in numbers adhering and changes in adhesion (slope) through the MRD were compared. There was a trend towards higher numbers adhering to the discs at the in-flow end of the MRD than at the outflow end, probably reflecting depletion of adherent bacteria in the interacting stream. Adhesion of Staph. epidermidis RP62A to siliconized glass and Teflon was reduced when compared to glass with increasing flow rates. Adhesion to stainless steel was not affected by flow rate and titanium gave a different slope of adhesion through the MRD when compared with glass, suggesting an interaction with different sub-populations within the interacting stream. Differences between siliconized glass and glass at flow rates of 300 ml h-1 were abolished by the addition of calcium or EDTA and reduced by the addition of magnesium. Increasing ionic strength reduced the statistical significance of the differences between glass and siliconized glass. Pre-conditioning of glass with pooled human plasma reduced adhesion compared with untreated glass and again gave a different slope to glass. The MRD linked to a chemostat can be used to compare directly bacterial adhesion to potential biomaterials. Variable depletion of the interacting stream should be taken into account in the interpretation of results. Divalent cation concentration, substrate properties and flow rate were important determinants of the comparative adhesion of Staph. epidermidis RP62A to surfaces.     

Composition,Production and Procurement of Glass at San Vincenzo al Volturno: An Early Medieval Monastic Complex in Southern Italy

136 glasses from the ninth-century monastery of San Vincenzo and its workshops have been analysed by electron microprobe in order to situate the assemblage within the first millennium CE glass making tradition. The majority of the glass compositions can be paralleled by Roman glass from the first to third centuries, with very few samples consistent with later compositional groups. Colours for trailed decoration on vessels, for vessel bodies and for sheet glass for windows were largely produced by melting the glass tesserae from old Roman mosaics. Some weakly-coloured transparent glass was obtained by re-melting Roman window glass, while some was produced by melting and mixing of tesserae, excluding the strongly coloured cobalt blues. Our data suggest that to feed the needs of the glass workshop, the bulk of the glass was removed as tesserae and windows from a large Roman building. This is consistent with a historical account according to which the granite columns of the monastic church were spolia from a Roman temple in the region. The purported shortage of natron from Egypt does not appear to explain the dependency of San Vincenzo on old Roman glass. Rather, the absence of contemporary primary glass may reflect the downturn in long-distance trade in the later first millennium C.E., and the role of patronage in the “ritual economy” founded upon donations and gift-giving of the time.     

Alteration of a Submarine Basaltic Glass under Environmental Conditions Conducive for Microorganisms: Growth Patterns of the Microbial Community and Mechanism of Palagonite Formation

In basaltic glass from the southern Mid-Atlantic-Ridge conducive environmental conditions for biogenic weathering resulted in excellent preserved microbial morphologies on glass surfaces. The distinct glass interface and open spaces between palagonite sheet and glass indicate a dissolution-reprecipitation mechanism of glass alteration potentially supported by microorganisms. On internal fracture surfaces, branching channels with widths at 20–30 μm containing longish structures with targeted dissolution of the glass by growing tips were observed. Alteration resulted in enrichment of Fe, Ti, P, and K in palagonite in amorphous mineral forms.     

Fungal biodeterioration of stained-glass windows

Biodeterioration of stained-glass windows by fungi was studied using historically accurate glass production methods. Glass reproductions were made according to the chemical composition determined by micro energy dispersive X-Ray Fluorescence of two historical glass windows belonging to King Ferdinand II's collection dating from the 15th and 17th centuries. Three distinct glasses compositions with different colours were selected and reproduced: i) a mixed-alkali colourless glass: ii) a purple potash-glass with manganese as chromophore, and iii) a brown potash-glass coloured by iron ions. The reproduced glass samples, with two initial surface morphologies (corroded and non-corroded), were inoculated with fungi previously isolated and identified on the original stained-glass windows as species of the genera Penicillium and Cladosporium. Physical and chemical glass surface alterations were analysed by means of optical microscopy, Raman microscopy, micro Infrared spectroscopy, and scanning electron microscopy with energy dispersive spectroscopy analysis. Results showed that fungi produced clear damage on all glass surfaces, present as spots and stains, fingerprints, biopiting, leaching and deposition of elements, and formation of biogenic crystals. Therefore, the inoculated fungi were able to biodeteriorate glasses with distinct compositions. Regarding the biodeterioration degree, there were no differences between the initial non-corroded and corroded glass surfaces.     

Gliding motility of Mycoplasma mobile can occur by repeated binding to N-acetylneuraminyllactose (sialyllactose) fixed on solid surfaces

Mycoplasma mobile relies on an unknown mechanism to glide across solid surfaces including glass, animal cells, and plastics. To identify the direct binding target, we examined the factors that affect the binding of Mycoplasma pneumoniae to solid surfaces and concluded that N-acetylneuraminyllactose (sialyllactose) attached to a protein can mediate glass binding on the basis of the following four lines of evidence: (i) glass binding was inhibited by N-acetylneuraminidase, (ii) glass binding was inhibited by N-acetylneuraminyllactose in a structure-dependent manner, (iii) binding occurred on glass pretreated with bovine serum albumin attached to N-acetylneuraminyllactose, and (iv) gliding speed depended on the density of N-acetylneuraminyllactose on glass.     

Late Byzantine mineral soda high alumina glasses from Asia Minor: a new primary glass production group

The chemical characterisation of archaeological glass allows the discrimination between different glass groups and the identification of raw materials and technological traditions of their production. Several lines of evidence point towards the large-scale production of first millennium CE glass in a limited number of glass making factories from a mixture of Egyptian mineral soda and a locally available silica source. Fundamental changes in the manufacturing processes occurred from the eight/ninth century CE onwards, when Egyptian mineral soda was gradually replaced by soda-rich plant ash in Egypt as well as the Islamic Middle East. In order to elucidate the supply and consumption of glass during this transitional period, 31 glass samples from the assemblage found at Pergamon (Turkey) that date to the fourth to fourteenth centuries CE were analysed by electron microprobe analysis (EPMA) and by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The statistical evaluation of the data revealed that the Byzantine glasses from Pergamon represent at least three different glass production technologies, one of which had not previously been recognised in the glass making traditions of the Mediterranean. While the chemical characteristics of the late antique and early medieval fragments confirm the current model of glass production and distribution at the time, the elemental make-up of the majority of the eighth- to fourteenth-century glasses from Pergamon indicate the existence of a late Byzantine glass type that is characterised by high alumina levels. Judging from the trace element patterns and elevated boron and lithium concentrations, these glasses were produced with a mineral soda different to the Egyptian natron from the Wadi Natrun, suggesting a possible regional Byzantine primary glass production in Asia Minor.     

Factors Affecting the Chemical Durability of Glass Used in the Pharmaceutical Industry

Delamination, or the generation of glass flakes in vials used to contain parenteral drug products, continues to be a persistent problem in the pharmaceutical industry. To understand all of the factors that might contribute to delamination, a statistical design of experiments was implemented to describe this loss of chemical integrity for glass vials. Phase I of this study focused on the effects of thermal exposure (prior to product filling) on the surface chemistry of glass vials. Even though such temperatures are below the glass transition temperature for the glass, and parenteral compounds are injected directly into the body, data must be collected to show that the glass was not phase separating. Phase II of these studies examined the combined effects of thermal exposure, glass chemistry, and exposure to pharmaceutically relevant molecules on glass delamination. A variety of tools was used to examine the glass and the solution contained in the vial including: scanning electron microscopy and dynamic secondary ion mass spectroscopy for the glass; and visual examination, pH measurements, laser particle counting, and inductively coupled plasma–optical emission spectrometry for the analysis of the solution. The combined results of phase I and II showed depyrogenation does not play a significant role in delamination. Terminal sterilization, glass chemistry, and solution chemistry are the key factors in the generation of glass flakes. Dissolution of silica may be an effective indicator that delamination will occur with a given liquid stored in glass. Finally, delamination should not be defined by the appearance of visible glass particulates. There is a mechanical component in the delamination process whereby the flakes must break away from the interior vial surface. Delamination should be defined by the observation of flakes on the interior surface of the vial, which can be detected by several other analytical techniques.     

A new heterobifunctional reagent for immobilization of biomolecules on glass surface

Synthesis of a new heterobifunctional reagent, [N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine] (NTMTA) is described for the immobilization of a variety of biomolecules on glass surface. Its triethoxysilyl group reacts with glass surface and trifluoroethanesulfonate ester structure reacts selectively with aminoalkyl/mercaptoalkyl function in biomolecules. The immobilization can be achieved by two ways involving two steps. The first route involves the reaction of NTMTA with glass beads followed by attachment of aminoalkyl- or mercaptoalkylated biomolecules. The second one involves the reaction of biomolecules, viz., oligonucleotides, proteins, etc., with NTMTA via their aminoalkyl or mercaptoalkyl functions to form a biomolecule conjugate, which is then reacted with glass beads (unmodified) to complete immobilization process. This has been demonstrated by successful immobilization of 5'-mercaptoalkyl- or aminoalkylated oligonucleotides and some commonly used enzymes on glass beads using NTMTA reagent.     

玻璃粉吸附核酸及其在植物核酸提取中应用的研究

为研究玻璃粉在植物核酸提取中的应用,比较了玻璃粉颗粒大小、离液盐种类及浓度、pH等条件对玻璃粉吸附核酸的影响,得出玻璃粉吸附核酸的各种最佳条件。结果表明,普通玻璃粉吸附核酸能力强于硅胶和硅藻土,玻璃粉颗粒的直径以83 μm为佳,pH 4.0时吸附效果达到最大。提取DNA时,NaCl浓度应大于3 mol/L,而提取RNA时,异硫氰酸胍大于2 mol/L就能取得很好的效果,此外,在玻璃粉吸附RNA前,需要加入50%以上的无水乙醇才能更好地吸附。利用玻璃粉制作简易纯化柱,可用于植物组织核酸提取纯化,所提取的核酸纯度高、完整性好,可用于酶切、杂交和PCR等实验。与传统方法相比,采用玻璃粉简易离心柱提取植物核酸,效果好、环保、快速、经济。     

Comparing cardiac action potentials recorded with metal and glass microelectrodes

Machine-pulled high-impedance glass capillary microelectrode is standard for transmembrane potential (TMP) recordings. However, it is fragile and difficult to impale, especially in beating myocardial tissues. We hypothesize that a high-impedance pure iridium metal electrode can be used as an alternative to the glass microelectrode for TMP recording. The TMPs were simultaneously recorded from isolated perfused swine right ventricles with a metal microelectrode and a standard glass microelectrode during pacing and during ventricular fibrillation. The basic morphology of TMP recorded with these electrodes was comparable. The action potential duration (APD) at 90% repolarization was 241 +/- 29 ms for the metal microelectrode and 236 +/- 31 ms for the glass microelectrode with a good correlation (r = 0.99, P < 0.0001). The maximum slope value of the APD restitution curves during pacing was also significantly correlated. One metal microelectrode and >20 glass microelectrodes were needed per study. We conclude that, in isolated perfused swine right ventricles, the TMP recorded by the metal microelectrode is comparable with that recorded by the glass microelectrode. Because the metal microelectrode is more durable than the glass microelectrode, it can serve as an alternative for APD recording and for restitution analyses.     

Immobilization of Chloroperoxidase on Aminopropyl-Glass

Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7.     

Whole Mounts of Small Embryos Attached Directly to Glass Slides

Suspensions of sea urchin embryos spread over the surface of glass slides were attached to the glass by a rapid coagulation of the surface with alcohol. This was done either by dipping the glass slides into absolute alcohol or by a short exposure to alcohol vapor. Thereafter the slides were immediately transferred to the fixative. A suitable procedure includes fixation with Carnoy's fluid (alcohol, chloroform, acetic acid; 6:3:1) and staining with Gomori's hematoxylin after acid hydrolysis.     

Characterization of oligodeoxyribonucleotide synthesis on glass plates

Achieving high fidelity chemical synthesis on glass plates has become increasingly important, since glass plates are substrates widely used for miniaturized chemical and biochemical reactions and analyses. DNA chips can be directly prepared by synthesizing oligonucleotides on glass plates, but the characterization of these micro-syntheses has been limited by the sub-picomolar amount of material available. Most DNA chip syntheses have been assayed using in situ coupling of fluorescent molecules to the 5′-OH of the synthesized oligonucleotides. We herein report a systematic investigation of oligonucleotide synthesis on glass plates with the reactions carried out in an automated DNA synthesizer using standard phosphoramidite chemistry. The analyses were performed using ³²P gel electrophoresis of the oligonucleotides cleaved from glass plates to provide product distribution profiles according to chain length of oligonucleotides. 5′-Methoxythymidine was used as the chain terminator, which permits assay of coupling reaction yields as a function of chain length growth. The results of this work reveal that a major cause of lower fidelity synthesis on glass plates is particularly inefficient reactions of the various reagents with functional groups close to glass plate surfaces. These problems cannot be detected by previous in situ fluorescence assays. The identification of this origin of low fidelity synthesis on glass plates should help to achieve improved synthesis for high quality oligonucleotide microarrays.     

Immobilization of oligonucleotides on glass surface using an efficient heterobifunctional reagent through maleimide-thiol combination chemistry

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl) cyclohexanamide (TPMC), was developed for the immobilization of thiol-modified oligonucleotides on an unmodified glass surface. The heterobifunctionality of the reagent was used for the construction of a DNA microarray in which the triethoxysilyl functionality has specificity toward a glass surface, whereas the maleimide functionality has thiol-modified oligonucleotides via a stable thioether linkage. Immobilization of DNA was achieved by two alternative approaches. In the first approach, the reagent TPMC was treated with oligonucleotides to get triethoxysilyl-oligonucleotide conjugate, which was then covalently attached via specific triethoxysilyl functionality to an unmodified glass surface. In the second approach, the reagent was first covalently linked with an unmodified glass surface to get maleimide functionality on a glass surface, which was then used for the immobilization of oligonucleotides via a stable thioether linkage. The applicability of the reagent was explored by hybridization studies with the fluorescein-labeled complementary DNA strand and in mismatch discrimination.     

蛋白质微阵列生产用琼脂糖修饰玻片制备的条件优化

目的:建立一种以琼脂糖修饰的玻片为载体的蛋白质微阵列制备的优化方法,比较琼脂糖修饰玻片和醛基修饰玻片及氨基修饰玻片对蛋白质固定效率的优劣。方法:将羊IgG固定在载体表面,经过洗涤、封闭,再加入Cy3标记的兔抗羊IgG,孵育,洗涤后用共聚焦激光扫描仪获取图像,检测各点的荧光强度,根据荧光强度确定最佳琼脂糖浓度,最佳NaIO4浓度,最佳固定时间以及封闭时间等实验条件。结果:琼脂糖浓度为1.2％、NaIO4浓度为20mmol／L、固定时间为1h、孵育时间为45min时,蛋白质在载体上的固定效率和反应活性最高。在固定的抗体浓度相同的情况下,琼脂糖修饰玻片荧光强度是醛基修饰玻片的2.6倍,是氨基修饰玻片的9倍。结论:确立了蛋白质微阵列生产用琼脂糖修饰玻片制备的优化条件,用该优化条件制备的琼脂糖玻片更适合用于蛋白质微阵列载体。     

Individual patterns of rhythmic swimming activity in Anguilla anguilla glass eels synchronised to water current reversal

The existence of distinct patterns of activity and swimming behaviour were tested in individual European glass eels by means of Bayesian inference mixture modelling. 36 glass eels were tagged using Visible Implant Elastomer and added to 36 untagged glass eels in February and April. Each group was presented with a change in water current direction every 6,2 h and videotaped during 2 weeks. Two hypotheses were tested: (i) all individuals display a similar pattern of behaviour within a tidal period, glass eels showing both positive and negative rheotaxis in opposite phase (M1 model) and (ii) individuals are distributed in two different groups, some glass eels swimming with and the others against the current (M2 model). Results showed that most glass eels displayed a positive or a negative rheotaxis and only a small number exhibited both behaviours. All swimming behaviours were synchronised to a change in current direction with a period close to the tidal one. Results are discussed in relation to synchronisation and migration behaviour.     

Potassium channel "inactivation" induced by soft-glass patch pipettes

We have studied the potassium currents of rat pituitary pars intermedia cells kept in primary culture using whole-cell recording with patch pipettes. The potassium current recorded with hard-glass pipettes is mainly carried by voltage-dependent channels that show slow inactivation in the presence of 0.5 mM internal EGTA. Fast "inactivation" of the potassium current is seen with patch pipettes fabricated from soft glass (soda glass or potash lead glass), and is probably caused by block of the potassium channels by di- or multivalent cations released from the glass.     

Adherence of streptococci to surface-modified glass

Four types of surface-modified glass were prepared. Aminopropyl glass was prepared by alkylsilylation of glass slides with gamma-aminopropyltriethoxysilane. This glass carries primary amino groups which may be protonated at pH 7.2. Owing to the presence of both positively charged ions and hydrophobic ethoxyl groups, the glass is considered to be amphipathic. Three other types of surface-modified glass slides were prepared from aminopropyl glass by forming Schiff's bases with three aldehydes: glucose, glyoxylic acid and hexanal. The aldehyde-treated slides were subsequently reduced using sodium borohydride. Thus, the surface of the glass was rendered hydrophilic, ampholytic or hydrophobic, respectively. The adherence of two Streptococcus sanguis strains and two Streptococcus mutans strains to the surface-modified glass slides was studied. Different strains showed differences in adherence to these slides depending on their physico-chemical surface properties. For S. sanguis ATCC 10556, hydrophobic bonds seemed to be most important, while in S. mutans OMZ 176, ionic interactions made the highest contribution to adhesion. Hydrogen bonds seemed to contribute least to adherence.     

Biodeteriorative processes on glass: experimental proof of the role of fungi and cyanobacteria

Biodeterioration of glass under the influence of fungi and cyanobacteria was simulated on model glasses produced according to the old recipes. Strains of fungi and cyanobacteria chosen for the investigation were isolated from biodeteriorated glass windows or similar indoor environment and are reported to be frequently involved in glass alteration. Growth of fungi and cyanobacteria resulted in the dense colonisation of the material with an expressed biofilm formation on the glass surface. The following deterioration phenomena were observed: micropitting and crack formation by all studied fungi and cyanobacteria; delineatingtraces of cells, hyphae and filaments on the glass surface; colour change of the surface due to fungal or cyanobacterial growth; biogenic minerals deposition as a consequence of the microbial metabolism on the glass surface. The pattern of glass biopitting produced in the experiment was very similar to the biopits observed on antique and medieval glasses (Krumbein et al., 1991). Crack formation pattern was strain-specific, but appeared to be independent of the chemical composition of the glass itself. The degree of deterioration was changing according to the sensitivity of the glass in question to corrosion.     

DNA微阵列基片的化学处理

对于合成后点样的DNA微阵列 ,基片的表面化学处理非常重要。它直接影响到样品与基片的结合效率 ,进而影响杂交结果。基片表面的各种化学修饰方法多种多样 ,物理吸附主要以赖氨酸包被为主。共价结合通常使用同源偶联分子或异源偶联分子 ,还可以在基片表面组装线状、分支状连接分子或包被琼脂糖。着重介绍了DNA微阵列的制备 ,即样品如何固定到玻璃基片上。总结了不同类型基片表面的化学修饰方法以及DNA与基片的化学结合。