Name: biofilm removal using carbon dioxide aerosols without nitrogen purge
Uploaded: 2016-11-06T15:00:00.000+00:00
Duration: 7 min 11 s
Description: Watch this Scientific Journal Video about Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge at JoVE.com

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning (# 2015R1A2A2A01006446).

1. Preparación de superficies para la formación de biofilm <ol><li> Cortar 1 mm de grosor, 304 placas de acero inoxidable en chips (10 × 10 mm 2) con un cortador mecánico. </li><li> Realizar la limpieza por ultrasonidos de los chips en acetona, metanol, y desionizada (DI) secuencialmente agua durante 10 min cada uno. Use un recipiente resistente a los disolventes, hecho de sustancias tales como vidrio, para eliminar la contaminación orgánica. </li><li> Enjuague los chips con un chorro de agua DI durante 3-5 seg. </li><li> Se secan las fichas utilizando el flujo de gas N2 durante 3-5 seg. </li></ol> 2. Preparación del cultivo bacteriano <ol><li> Tome una P. putida KT2440 (proporcionado amablemente por el Prof. Sung Kuk Lee, UNIST, Corea del Sur) stock almacenado en un -80 ° C congelador. </li><li> Descongele a temperatura ambiente después de 1 min, y la capa superior de la solución madre congelada se convierte en aguanieve. Sumergir un bucle en la capa derretida de la solución madre. </li><li> Utilice este bucle a la raya a las bacterias en un medio Luria-Bertani placa (LB) que contiene 1,5% de agar. </li><li> Incubar la placa durante la noche a 30 ° C para crecer las colonias bacterianas. </li><li> Elija una única colonia de la placa usando un bucle fresco. </li><li> Inocular 10 ml de caldo LB en un tubo cónico de 50 ml con el bucle que contiene la colonia bacteriana única. </li><li> Incubar el caldo en una incubadora de agitación durante 16 horas a 30 ° C y 160 rpm. </li></ol> 3. La formación de biopelículas en la Superficie <ol><li> Recoger cada una de las fichas preparadas con unas pinzas y sumergirlos en un 70% de etanol 5 veces durante 1-2 segundos cada uno, para esterilizar la superficie de cada chip. Asegúrese de que las virutas se llevan a cabo con pinzas durante la inmersión. </li><li> Sumergir cada chip en agua DI autoclave y en LB caldo de forma secuencial, 5 veces durante 1-2 segundos cada uno, para eliminar el etanol restante. </li><li> Colocar estos chips en placas de cultivo de 6 pocillos con 2 chips y 5 ml de caldo LB por pocillo. </li><li> Diluir el cultivo bacteriano hasta que la concentración en los tramos caldo LB8 × 10 8 células / ml (densidad óptica a la longitud de onda 600 nm: ~ 0,8). </li><li> Inocular cada pocillo con 50 l de cultivo bacteriano diluido. </li><li> Incubar las placas a 30 ° C sin agitación durante 24 horas para la formación de biopelículas. </li></ol> 4. Limpieza de CO 2 en aerosol <ol><li> Dip los chips de la biopelícula formada en tampón acetato de amonio 10 mM (volátiles) 5 veces para eliminar débilmente unidos y bacterias planctónicas. </li><li> Seque estos chips en una cabina de seguridad biológica, donde el aire fluye suavemente. </li><li> Inmediatamente después del secado, colocar un chip en el lugar de carga, que es de 20 mm de la boquilla de CO2 a lo largo del eje del chorro. Incline el eje del chorro a un ángulo de 40 °. </li><li> Establecer las presiones de estancamiento de CO2 y N2 gaseoso a 5,3 MPa y 0,7 MPa, respectivamente, utilizando los reguladores de presión de gas. </li><li> Aplicar el chorro de aerosol en la parte central del chip. Aerosoles blancos incluyendo el CO2 sólido debese visible. &quot;Encender&quot; la válvula de solenoide para el CO 2 durante 5 segundos, y luego convertirlo en &quot;off&quot; durante 3 segundos (ciclo: 8 seg) periódicamente con un interruptor controlado manualmente. Si una purga de nitrógeno tiene que ser utilizado, a su vez en la válvula de solenoide para un suministro continuo de N 2. </li><li> Tratar los chips con CO 2 aerosoles para 16, 40, y 88 seg con y sin purgas de nitrógeno. Conservar los chips sin tratamiento como controles negativos. </li></ol> 5. Análisis de eficacia de la eliminación <ol><li> Preparar 1 M mancha de ácido nucleico de fluorescencia verde (longitud de onda de excitación / emisión: 480/500 nm) en agua DI para teñir las células bacterianas en el control y patatas fritas tratadas con aerosol. </li><li> Coloque las fichas en la solución de tinción. </li><li> Se incuban las fichas en una incubadora sin luz a 37 ° C durante 30 minutos. </li><li> Después de la incubación, lavar suavemente las virutas con un chorro de agua DI para eliminar el exceso de colorante fluorescente. </li><li> Se seca el wi fichasTH flujo de gas N 2. </li><li> Tome fluorescencia imágenes microscópicas de 5 campos aleatorios de vista (321 × 240 m 2) para cada chip utilizando un microscopio de epifluorescencia, una lente objetivo de 40X, y una cámara CCD. </li><li> Obtener la intensidad de fluorescencia para cada imagen utilizando un software de procesamiento de imágenes tales como ImageJ. En ImageJ, utilizar la función de &quot;Antecedentes Reste&quot; en el menú &quot;Acción&quot;, y seleccione &quot;densidad integrado&quot; en la ventana &quot;Establecer medidas&quot; en el menú &quot;Analizar&quot;. Hacer el &quot;medición&quot; en el menú &quot;Analizar&quot; para obtener la intensidad de fluorescencia. </li><li> Calcular la eficiencia de eliminación de biofilm de acuerdo con la fórmula siguiente: 100% x (I de chips de control - I de virutas tratadas) / (I de chips de control), donde I es la intensidad de la fluorescencia calculada. </li><li> Obtener el promedio de las eficiencias de eliminación y las desviaciones estándar. Use por lomenos 4 chips para cada caso. </li></ol>

<ol>
	<li>Jain, A., Gupta, Y., Agrawal, R., Khare, P., Jain, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms+-+A+microbial+life+perspective:+A+critical+review.">Biofilms - A microbial life perspective: A critical review.</a> Crit. Rev. Ther. Drug. 24 (5), 393-443 (2007).</li><li>Bott, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofouling+control+with+ultrasound.">Biofouling control with ultrasound.</a> Heat Transfer Eng. 21 (3), 43-49 (2000).</li><li>Meyer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+prevention,+removal+and+killing+of+biofilms.">Approaches to prevention, removal and killing of biofilms.</a> Int. Biodeterior. Biodegradation. 51 (4), 249-253 (2003).</li><li>Hong, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+electric+currents+on+bacterial+detachment+and+inactivation.">Effect of electric currents on bacterial detachment and inactivation.</a> Biotechnol. Bioeng. 100 (2), 379-386 (2008).</li><li>Nandakumar, K., Obika, H., Utsumi, A., Ooie, T., Yano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+laser+ablation+of+laboratory+developed+biofilms+using+an+Nd:YAG+laser+of+532+nm+wavelength.">In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength.</a> Biotechnol. Bioeng. 86 (7), 729-736 (2004).</li><li>Gibson, H., Taylor, J. H., Hall, K. E., Holah, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+cleaning+techniques+used+in+the+food+industry+in+terms+of+the+removal+of+bacterial+biofilms.">Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms.</a> J. Appl. Microbiol. 87 (1), 41-48 (1999).</li><li>Kang, M. Y., Jeong, H. W., Kim, J., Lee, J. W., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+biofilms+using+carbon+dioxide+aerosols.">Removal of biofilms using carbon dioxide aerosols.</a> J. Aerosol Sci. 41 (11), 1044-1051 (2010).</li><li>Cha, M., Hong, S., Kang, M. Y., Lee, J. W., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas-phase+removal+of+biofilms+from+various+surfaces+using+carbon+dioxide+aerosols.">Gas-phase removal of biofilms from various surfaces using carbon dioxide aerosols.</a> Biofouling. 28 (7), 681-686 (2012).</li><li>Dwidar, M., Hong, S., Cha, M., Jang, J., Mitchell, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+application+of+bacterial+predation+and+carbon+dioxide+aerosols+to+effectively+remove+biofilms.">Combined application of bacterial predation and carbon dioxide aerosols to effectively remove biofilms.</a> Biofouling. 28 (7), 671-680 (2012).</li><li>Cha, M., Hong, S., Lee, S. Y., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+different-age+biofilms+using+carbon+dioxide+aerosols.">Removal of different-age biofilms using carbon dioxide aerosols.</a> Biotechnol. Bioprocess Eng. 19 (3), 503-509 (2014).</li><li>Singh, R., Monnappa, A. K., Hong, S., Mitchell, R. J., Jang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Carbon+Dioxide+Aerosols+on+the+Viability+of+Escherichia+coli+during+Biofilm+Dispersal.">Effects of Carbon Dioxide Aerosols on the Viability of Escherichia coli during Biofilm Dispersal.</a> Sci. Rep. 5, 13766 (2015).</li><li>Sherman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+Dioxide+Snow+Cleaning.">Carbon Dioxide Snow Cleaning.</a> Particul. Sci.Technol. 25 (1), 37-57 (2007).</li></ol>

The authors declare that they have no competing financial interests.

Previously, we conducted optimization studies on CO2 gas pressure, jet angle, and distance to the solid surface in CO2 aerosol cleaning7. Unlike our previous studies, in the present study, a nitrogen purge was not included in the aerosol (Figure 1). Moreover, 304 stainless steel was used in this protocol, since it is one of the most common stainless steels and is widely used in the food industry. The polished surface is beneficial for fluorescence analysis because of a un...

Biofilms are complex bacterial community structures in which bacterial cells are embedded within self-produced matrices of extracellular polymeric substances, held together, and protected from the external environment. Biofilms can present a public health risk and cause economic losses because they can form on various surfaces, including medical implant materials and devices, food processing equipment, and heat exchangers. In fact, biofilms have been found to be associated with 65% of all bacterial infectious diseases in humans, according to the Centers for Disease Control and Prevention1.
Autoclaves and disinfectants such as chlorine have generally been used for the inactivation of biofilms. However, the use of an autoclave is limited for surfaces that can neither withstand high temperature steam nor be placed into the autoclave. Disinfectants are not suitable for surfaces sensitive to chemical treatment or prone to collecting toxic oxidation products2. In addition, the biofilm should not only be inactivated, but also removed in order to prevent the attachment of new cells onto the surface, thereby forming a new biofilm3. However, it is difficult to remove biofilms using methods based on viscous fluid shear because the flow velocity near the surface is almost zero and the shear force usually cannot overcome the adhesive forces of micron- and submicron-sized substances. Moreover, the biofilm matrix is known to act as a physical and chemical barrier1.
Many physical and mechanical techniques have been developed to remove biofilms from surfaces, including ultrasonic vibration1, electric currents4, laser irradiation5, and high-pressure water sprays6. Each technique has its own pros and cons. Ultrasonic vibration and electric current can be used to control biofilm formation; however, they require a particular configuration and conductive surfaces, respectively, requiring additional shear stress1, 4. Laser irradiation can be applied to a limited area and to hard surfaces; however, some live and dead cells remain on the surface5. High-pressure water sprays effectively remove biofilms; however, their high momentum can cause damage to soft substrates6.
Biofilm removal using CO2 aerosols has been previously proposed. It has shown promising results, with high removal efficiencies within a short time7-11. CO2 aerosols are generated by adiabatic expansion of a high-pressure CO2 gas through a nozzle, and they are applied to the surfaces contaminated with a biofilm. This cleaning technique utilizes the momentum transfer of solid CO2 particles and the solvent action of the melted CO2 liquids, followed by the aerodynamic shear force of the CO2 gas12. Compared with high-pressure N2 gas jets, CO2 aerosol jets at the same stagnation pressure are much more effective in removing E. coli biofilms7. Moreover, although the momentum of the solid CO2 that is delivered to the bacteria is considerably high, the momentum of the total aerosol jet applied to the solid surface is substantially lower than that of water jets. Therefore, damage-free cleaning is possible using this CO2 aerosol technique.
In this protocol, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from a polished stainless steel surface. In fact, nitrogen purges have been used in many CO2 aerosol studies to increase the removal efficiency by reducing the moisture condensation generated during the cleaning, even though heating with a hot plate or infrared lamps and employing dry boxes have also been adopted12. The surface biofilm formation and the optimized cleaning procedures are described below. The removal efficiency was evaluated by measuring the fluorescence intensity of the biofilm on the surface.

<table><tbody><tr><td>304 stainless steel</td><td>Steelni&lt;br/&gt; (South Korea)</td><td></td><td>Polished and diced ones</td></tr><tr><td>Ultrasonic cleaner</td><td>Branson&lt;br/&gt; (USA)</td><td>5510E-DTH</td><td></td></tr><tr><td>Luria-Bertani (LB)</td><td>Becton, Dickinson and Company&lt;br/&gt; (USA)</td><td>244620</td><td>500 g</td></tr><tr><td>Agar</td><td>Becton, Dickinson and Company&lt;br/&gt; (USA)</td><td>214010</td><td>500 g</td></tr><tr><td>6-well culture plate</td><td>SPL Life Sciences&lt;br/&gt; (South Korea)</td><td>32006</td><td></td></tr><tr><td>Ammonium acetate buffer</td><td>Sigma-Aldrich&lt;br/&gt; (USA)</td><td>667404</td><td>10 mM</td></tr><tr><td>Dual gas unit</td><td>Applied Surface Technologies&lt;br/&gt; (USA)</td><td>K6-10DG</td><td>One nozzle for CO&lt;sub&gt;2&lt;/sub&gt; gas&lt;br/&gt; &amp;amp; 8 nozzles for N&lt;sub&gt;2&lt;/sub&gt; gas</td></tr><tr><td>SYTO9</td><td>Thermo Fissher Scientific&lt;br/&gt; (USA)</td><td>Invitrogen</td><td>Excitaion: 480 nm&lt;br/&gt; Emission: 500 nm</td></tr><tr><td>Epifluorescence microscope&amp;nbsp;</td><td>Nikon (Japan)</td><td>Eclipse 80i</td><td></td></tr><tr><td>40X objective lens</td><td>Nikon&lt;br/&gt; (Japan)</td><td>Plan Fluor</td><td>NA: 0.75</td></tr><tr><td>CCD camera&amp;nbsp;</td><td>Photometrics&lt;br/&gt; (USA)</td><td>Cool SNAP HQ2</td><td>Monochrome</td></tr></tbody></table>

biofilm removal using carbon dioxide aerosols without nitrogen purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p &gt; 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

CO 2 aerosoles se utilizan para eliminar el P. putida biofilms de SUS304 superficies (Figura 1). La mayor parte de las superficies estaban cubiertas por el biofilm después de 24 horas de crecimiento. La mayor parte de la biopelícula se retiró usando el CO 2 aerosoles (Figura 2). Como era de esperar, la Figura 3 muestra un aumento de la eficiencia de eliminación de biofilm como el tiempo de tratamiento aerosol CO 2 aumentado....

Watch this Scientific Journal Video about Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge at JoVE.com

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

La eliminación del biofilm El uso de aerosoles de dióxido de carbono sin purga de nitrógeno

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

biofilm-removal-using-carbon-dioxide-aerosols-without-nitrogen-purge

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6.3K vistas.  Ulsan National Institute of Science and Technology (UNIST). Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Video: Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Los modelos de co-cultivo de Pseudomonas aeruginosa Biofilms Cultivado en las células vivas las vías respiratorias humanas

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Investigación

Inmunología e Infección

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Preparación de hidrófobos orgánicos marcos de metal a través de Plasma Enhanced Chemical Vapor Deposition de perfluoroalcanos para la eliminación de amoniaco

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Química

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

Preparación de biopolímeros aerogeles el uso de disolventes verdes

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

Non-equilibrium Microwave Plasma for Efficient High Temperature Chemistry

A flowing microwave plasma based methodology for converting electric energy into internal and/or translational modes of stable molecules with the purpose of efficiently driving non-equilibrium chemistry is discussed. The advantage of a flowing plasma reactor is that continuous chemical processes can be driven with the flexibility of startup times in the seconds timescale. The plasma approach is generically suitable for conversion/activation of stable molecules such as CO2, N2 and CH4. Here the reduction of CO2 to CO is used as a model system: the complementary diagnostics illustrate how a baseline thermodynamic equilibrium conversion can be exceeded by the intrinsic non-equilibrium from high vibrational excitation. Laser (Rayleigh) scattering is used to measure the reactor temperature and Fourier Transform Infrared Spectroscopy (FTIR) to characterize in situ internal (vibrational) excitation as well as the effluent composition to monitor conversion and selectivity.

This article describes a flowing microwave reactor that is used to drive efficient non-equilibrium chemistry for the application of conversion/activation of stable molecules such as CO2, N2 and CH4. The goal of the procedure described here is to measure the in situ gas temperature and gas conversion.

Plasma de microondas sin equilibrio para una química eficiente a altas temperaturas

A flowing microwave plasma based methodology for converting electric energy into internal and/or translational modes of stable molecules with the purpose ...

Precision Milling of Carbon Nanotube Forests Using Low Pressure Scanning Electron Microscopy

A nanoscale fabrication technique appropriate for milling carbon nanotube (CNT) forests is described. The technique utilizes an environmental scanning electron microscope (ESEM) operating with a low pressure water vapor ambient. In this technique, a portion of the electron beam interacts with the water vapor in the vicinity of the CNT sample, dissociating the water molecules into hydroxyl radicals and other species by radiolysis. The remainder of the electron beam interacts with the CNT forest sample, making it susceptible to oxidation from the chemical products of radiolysis. This technique may be used to trim a selected region of an individual CNT, or it may be used to remove hundreds of cubic microns of material by adjusting ESEM parameters. The machining resolution is similar to the imaging resolution of the ESEM itself. The technique produces only small quantities of carbon residue along the boundaries of the cutting zone, with minimal effect on the native structural morphology of the CNT forest.

Low pressure scanning electron microscopy in a water vapor ambient is used to machine nanoscale to microscale features in carbon nanotube forests.

Fresado de precisión de nanotubos de carbono de los bosques El uso de baja presión de barrido Microscopía Electrónica

A nanoscale fabrication technique appropriate for milling carbon nanotube (CNT) forests is described. The technique utilizes an environmental scanning ...

Ingeniería

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are encapsulated within the aerogels. However, few reports on encapsulating proteins within sol&#8211;gels that are processed to form aerogels exist. A procedure for encapsulating cytochrome c (cyt. c) in silica (SiO2) sol-gels that are supercritically processed to form bioaerogels with gas-phase activity for nitric oxide (NO) is presented. Cyt. c is added to a mixed silica sol under controlled protein concentration and buffer strength conditions. The sol mixture is then gelled and the liquid filling the gel pores is replaced through a series of solvent exchanges with liquid carbon dioxide. The carbon dioxide is brought to its critical point and vented off to form dry aerogels with cyt. c encapsulated inside. These bioaerogels are characterized with UV-visible spectroscopy and circular dichroism spectroscopy and can be used to detect the presence of gas-phase nitric oxide. The success of this procedure depends on regulating the cyt. c concentration and the buffer concentration and does not require other components such as metal nanoparticles. It may be possible to encapsulate other proteins using a similar approach making this procedure important for potential future bioanalytical device development.

Encapsulating Cytochrome c in Silica Aerogel Nanoarchitectures without Metal Nanoparticles while Retaining Gas-phase Bioactivity

This procedure describes how to encapsulate cytochrome c (cyt. c) in silica (SiO2) sol-gels, process these gels to form bioaerogels, and use these bioaerogels to rapidly recognize nitric oxide (NO) through a gas-phase reaction. This type of protocol may aid in the future development of biosensors or other bioanalytical devices.

encapsular citocromo<em&gt; c</em&gt; El aerogel de sílice en nanoarquitecturas sin metal nanopartículas Mientras Retienen bioactividad en fase gaseosa

Applications such as sensors, batteries, and fuel cells have been improved through the use of highly porous aerogels when functional compounds are ...

Bioingeniería

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy. Quorum-quenching lactonases have been reported to be effective disrupters of quorum-sensing circuits. However, there have been very few reports of the effective use of these enzymes in disrupting bacterial biofilm formation. This protocol describes a method to disrupt biofilm formation in a clinically relevant A. baumannii S1 strain through the use of an engineered quorum-quenching lactonase. Acinetobacter baumannii is a major human pathogen implicated in serious hospital-acquired infections globally and its virulence is attributed predominantly to its biofilm's tenacity. The engineered lactonase treatment achieved significant A. baumannii S1 biofilm reduction. This study also showed the possibility of using engineered quorum-quenching enzymes in future treatment of biofilm-mediated bacterial diseases. Lastly, the method may be used to evaluate the competency of promising quorum-quenching enzymes.

Quorum-quenching enzymes are anti-virulent and anti-bacterial options that can mitigate pathogenesis without risk of incurring resistance, by preventing the expression of virulence factors and genes associated with antibiotic resistance and biofilm formation. In this study, we report a method that demonstrates the efficacy of quorum-quenching enzymes in bacterial biofilm disruption.

Interrupción Anti-virulenta de patógenos biopelículas utilizando Engineered Quórum para saciar lactonasas

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologías para el estudio<em&gt; B. subtilis</em&gt; Las biopelículas como modelo para la caracterización de Pequeño Biofilm inhibidores de moléculas

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural integrity. The nosocomial pathogen, Pseudomonas aeruginosa, is known to adopt a biofilm mode of growth, causing chronic pulmonary infection in patients with cystic fibrosis (CF). The computer program, COMSTAT, is a useful tool for quantifying antimicrobial-induced changes in P. aeruginosa biofilm architecture by extracting data from three-dimensional confocal images. However, standardized operation of the software is less commonly addressed, which is important for optimal reporting of biofilm behavior and cross-center comparison. Thus, the aim of this protocol is to provide a simple and reproducible framework for quantifying in vitro biofilm structures under varying antimicrobial conditions via COMSTAT. The technique is modeled using a CF P. aeruginosa isolate, grown in the form of biofilm replicates, and exposed to tobramycin and the anti-Psl monoclonal antibody, Psl0096. The step-by-step approach aims to reduce user ambiguity and minimize the chance of overlooking crucial image-processing steps. Specifically, the protocol emphasizes the elimination of subjective variations associated with the manual operation of COMSTAT, including image segmentation and the selection of appropriate quantitative analysis functions. Although this method requires users to spend additional time processing confocal images prior to running COMSTAT, it helps minimize misrepresented biofilm heterogenicity in automated outputs.

Quantifying the Effects of Antimicrobials on In vitro Biofilm Architecture using COMSTAT Software

Antimicrobial-induced changes to Pseudomonas aeruginosa biofilm architecture differ among clinical isolates cultured from patients with cystic fibrosis and chronic pulmonary infection. Following confocal microscopy, COMSTAT software can be utilized to quantify variations in biofilm architecture (e.g., surface area, thickness, biomass) for individual isolates to assess the efficacy of anti-infective agents.

Cuantificación de los efectos de los antimicrobianos en la arquitectura de biopelículas in vitro mediante el software COMSTAT

Biofilms are aggregates of microorganisms that rely on a self-produced matrix of extracellular polymeric substance for protection and structural ...

<meta charset="utf8"></head><body>Modelo CF adaptable para pruebas de antimicrobianos y análisis de dinámica microbiana

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

<meta charset="utf8"></head><body>Desarrollo de un modelo de biofilm de colonias polimicrobianas para probar antimicrobianos en la fibrosis quística

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Desarrollo de un modelo de biofilm de colonias polimicrobianas para probar antimicrobianos en la fibrosis quística

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

La eliminación del biofilm El uso de aerosoles de dióxido de carbono sin purga de nitrógeno

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