Biopelícula relacionadas con el trabajo en el laboratorio es financiado por donaciones y numeradas R21DE017294 R21AI080930 del Instituto Nacional de Investigación Dental y Craneofacial y el Instituto Nacional de Alergias y Enfermedades Infecciosas (a JLL-R.). El contenido es responsabilidad exclusiva de sus autores y no representan necesariamente la opinión oficial de la NIDCR, el NIAID oa los NIH.

1. Preparación de C. albicans C. albicans es un microorganismo del grupo de riesgo 1/BSL1. Recuerde siempre que un buen uso de las técnicas de asepsia / estéril para trabajar con este microorganismo y seguir los procedimientos institucionales para la eliminación apropiada de tales materiales. <ol><li> Preparar un cultivo de una noche de C. albicans en YPD (levadura peptona dextrosa) medio líquido mediante la inoculación de una sola colonia de C. albicans en 25 ml de YPD. </li><li> Incubar la cultura en un agitador orbital (180 rpm) a 30 ° C durante la noche. La mayoría de C. albicans cepas crecerá a medida que las células de levadura bajo estas condiciones. </li><li> Centrifugar los cultivos durante la noche (alrededor de 3.000 rpm, 5-10 minutos), lavar las células dos veces con PBS estéril, y resuspender el precipitado final en unos 20-25 ml de medio RPMI 1640 tamponado con 165 mM de ácido morpholinepropanesulfonic a pH 7,0 y pre-calentado a 37 ° C (a partir de ahora este medio se conoce simplemente como &quot;RPMI 1640&quot;). </li><li> Recuento de células con un hemocitómetro. Después de contar, preparar una suspensión de células a una densidad final de 1,0 x 10 6 células / ml en RPMI 1640. Nota: Dado que las células tienen una tendencia a agregarse, es importante agitar vigorosamente entre los lavados y antes de pipetear. </li></ol> 2. Instalación de la placa de 96 pocillos de microtitulación para la formación del biofilm <ol><li> Con una pipeta multicanal añadir 100 ml de C. albicans suspensión en los pozos seleccionados de la placa de 96 pocillos de microtitulación (-s). No agregar celdas a los pozos en la columna 12, ya que estos sirven como controles negativos. </li><li> Cubrir la placa de microtitulación de todo con su tapa original, sellado con parafilm, colocarlo dentro de una incubadora y se incuba durante 24 horas a 37 ° C. La duración de la incubación se puede adaptar al diseño experimental específico. Por ejemplo, es posible examinar la cinética de formación del biofilm en un período de 24 a 48 horas en la siembra y el procesamiento de múltiples placas de cada plato en diferentes puntos temporales (es decir, 2, 4, 8, 12, 24 y 48 horas) </li><li> Después de la formación del biofilm, usando una pipeta multicanal aspirar el medio con cuidado de no tocar y alterar las biopelículas que se forman en cada uno de los pozos. </li><li> Utilizando una pipeta multicanal placas se lavan tres veces en PBS estéril (200 l por pocillo). Por otra parte, el uso de una lavadora automática de placas de microtitulación. Entre los mismos, y sobre todo después del último lavado, la fuga de las placas en posición invertida por secante con toallas de papel para quitar cualquier residuo de PBS. En este punto, los biofilms formados en el fondo de los pozos deben ser claramente visibles incluso a simple vista y también se puede visualizar mediante un microscopio invertido (Figura 1). Los biofilms son ahora listo para ser procesado por las pruebas de sensibilidad antifúngica ensayos. (Si el objetivo principal del experimento es evaluar el grado de formación del biofilm, las placas están listas para ser procesadas por el método colorimétrico. Para ello, el reactivo XTT / menadiona (ver paso 3) se pueden agregar y el color resultante leer con un lector de placas de microtitulación). </li></ol> 3. Pruebas de sensibilidad antifúngica de las biopelículas <ol><li> A partir de soluciones madre o el polvo, preparar una solución de trabajo en medio RPMI 1640 de cada uno de antimicóticos para ser probado. Las concentraciones típicas de alta son 1.024 mg / ml para fluconazol, y 16 mg / ml tanto para la anfotericina B y caspofungina. Otras concentraciones pueden ser utilizados para los diferentes agentes. </li><li> Con una pipeta multicanal, 200 l de la alta concentración de trabajo de antimicóticos para los pozos correspondientes en la columna 1 de cada placa de microtitulación que contienen las biopelículas fúngicas. </li><li> Añadir 100 ml de RPMI 1640 a cada pocillo de las columnas 2 a 10. </li><li> Añadir 100 ml de RPMI 1640 a los pozos en la columna 11. </li><li> Eliminar 100 L de agente antifúngico de los pozos de la columna 1 y añadir a los pozos adyacentes en la columna 2 (ya que contiene 100 l de media). </li><li> Mezclar el contenido y pipeteando arriba y abajo para realizar una dilución doble de serie, y eliminar las puntas. </li><li> Repita el movimiento a la derecha hasta los pozos de la columna 10, después de lo cual se descarta la final de 100 l de volumen de los pozos de la columna 10 después de la mezcla. De esta manera, una serie de diluciones al doble de su agente (-s) de interés se han creado a partir de la mayoría concentrados en los pozos de la columna 1, por lo que se concentró en los pozos de la columna 10. Biofilms sin respuesta en la columna 11 servirán como controles positivos, y los pozos se agotan en la columna 12 servirán como controles negativos. </li><li> Se cubren las placas con sus tapas, sello con parafilm y se incuba durante 24 -48 horas a 37 ° C. </li><li> Después del período de incubación, lavar los platos como en el Paso 2.4 antes (3 x PBS). </li><li> Con una pipeta multicanal añadir 100 ml de XTT / menadiona solución a cada pocillo que contenga un biofilm pre-lavado, así como a los pozos de control negativo para la medición de la X de fondoTT-colorimétrico los niveles. <ol><li> La XTT se prepara como una solución saturada a 0,5 g / L de lactato de Ringer estéril, PBS o solución salina, que debe ser esterilizada por filtración, con un 0,22 micras de poro del filtro de tamaño. La solución XTT es sensible a la luz, por lo que se debe cubrir con papel de aluminio durante la preparación. Una vez preparada y esterilizada por filtración, alícuota en 10 ml de volumen de trabajo, y guardar a -70 ° C. Proteger los tubos de la luz con papel de aluminio. Descongele sólo como tubos que sean necesarias para un experimento en particular antes de su uso. </li><li> Menadiona se prepara como una solución de 10 mM de valores en acetona al 100%, alícuotas en volúmenes más pequeños (unos 50 l) y almacenados a -70 ° C. Prepare la solución XTT / menadiona antes de su uso, mediante la adición de 1 l de la solución madre de menadiona con un tubo que contiene 10 ml de la solución XTT descongelado. </li></ol></li><li> Se cubren las placas en papel de aluminio y se incuban en la oscuridad durante 2 horas a 37 ° C. </li><li> Descubrir las placas. Con una pipeta multicanal eliminar el 80 l del sobrenadante resultante de color de cada pozo y la transferencia en los pocillos correspondientes de una nueva placa de microtitulación. </li><li> Leer la placa (-s) en un lector de placas microtiter a 490 nm. </li><li> Calcular la concentración inhibitoria mínima sésiles SMIC50 y SMIC80, que son las concentraciones de antifúngicos en el que se detecta una disminución del 50% o 80% en las lecturas colorimétricas en comparación con el control de biofilms formados en la ausencia de medicamentos antifúngicos (en este caso los valores de la columna 11 , recuerda también a restar los valores de los controles negativos de los pozos en la columna 12 contiene sólo XTT). Resultados SMIC se puede presentar como una tabla (es decir, múltiples aislamientos contra varios antifúngicos) o, alternativamente, los resultados de cada individuo aislado de hongos contra hongos pueden ser presentados en forma de gráfico mediante el trazado de la inhibición por ciento frente a la concentración de antimicóticos. </li></ol> Ejemplo: Después de restar los valores en el control negativo, la densidad óptica promedio de las biopelículas de control formado en la columna 11 es 1,32. El SMIC50 es la concentración más baja antifúngico que conduce a la reducción de&gt; 50% en las lecturas colorimétricas, en este caso menos de 1,32 x 50/100 = 0,66. Asimismo, el SMIC80 es la concentración más baja antifúngico con lo que disminuye&gt; 80% en las lecturas colorimétricas, en este caso menos de 1,32 x 20/100 = 0.264. 4. Resultados representante La figura 1 muestra una microfotografía de una C. albicans formación de biopelículas sobre el fondo de un pozo en una placa de microtitulación de 96 pocillos tomadas con un microscopio invertido. La figura 2 muestra XTT-colorimétrico lecturas (OD 490 valores) para cada una de las biopelículas de 11 C. albicans cepa de tipo salvaje formado en cada una de las 8 líneas diferentes de la misma placa de 96 pocillo de microtitulación. La Figura 3 muestra la actividad de la anfotericina B en diferentes concentraciones en contra de C. albicans biopelículas, las flechas indican SMIC50 y SMIC80 valores. <img src="/files/ftp_upload/2287/2287fig1.jpg" alt="Figura 1" /> Figura 1. (A) El panel A muestra una microfotografía tomada con una cámara conectada a un microscopio invertido de una C. albicans formación de biopelículas sobre el fondo del pozo después de la aspiración de medio RPMI y posteriores lavados con PBS. (B) Una micrografía de una típica C. albicans biofilm visualiza mediante microscopía electrónica de barrido. Las barras son 100 micras y 10 micras para los grupos A y B, respectivamente. <img src="/files/ftp_upload/2287/2287fig2.jpg" alt="Figura 2" /> Figura 2. Formación de múltiples equivalentes C. albicans biopelículas en placas de 96 pocillos de microtitulación. lecturas colorimétricas (OD 490 valores) de XTT de reducción de los ensayos de los biofilms formados por una C. albicans de tipo salvaje en los pozos de placas de microtitulación. Los valores son para el 11 de biofilms independiente formada en cada uno de 8 líneas diferentes de la misma placa de 96 pocillo de microtitulación. Los resultados de las diferentes filas se compararon mediante análisis de un solo sentido de varianza y el test de Bartlett para homogeneidad de varianzas y comparaciones múltiples de Bonferroni el post-test. No se observaron diferencias estadísticamente significativas se observaron al comparar todos los pares de filas entre sí (P&gt; 0,05). <img src="/files/ftp_upload/2287/2287fig3.jpg" alt="Figura 3" /> Figura 3. Los resultados típicos de las pruebas de sensibilidad antifúngica contra C. albicans biofilms. Gráfico que muestra los resultados típicos de la eficacia de diferentes concentraciones de anfotericina B contra las biopelículas de C. albicans cepa de tipo salvaje. Los valores se expresan como porcentaje medio de lecturas colorimétricas para los ensayos de reducción de XTT-en comparación con el control de los pozos. SMIC50 y SMIC80 valores se indican con flechas.

<ol>
	<li>Bachmann, S. P. <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+activity+of+caspofungin+against+Candida+albicans+biofilms.">In vitro activity of caspofungin against Candida albicans biofilms.</a> Antimicrob Agents Chemother. 46, 3591-3596 (2002).</li><li>Hawser, S. P., Norris, H., Jessup, C. J., Ghannoum, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+a+2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-t+etrazolium+hydroxide+(XTT)+colorimetric+method+with+the+standardized+National+Committee+for+Clinical+Laboratory+Standards+method+of+testing+clinical+yeast+isolates+for+susceptibility+to+antifungal+agents.">Comparison of a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-t etrazolium hydroxide (XTT) colorimetric method with the standardized National Committee for Clinical Laboratory Standards method of testing clinical yeast isolates for susceptibility to antifungal agents.</a> J Clin Microbiol. 36, 1450-1452 (1998).</li><li>Kuhn, D. M., Balkis, M., Chandra, J., Mukherjee, P. K., Ghannoum, M. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+albicans+transcription+factor+Bcr1p:+a+regulator+of+cell-surface+genes+and+biofilm+formation.">C. albicans transcription factor Bcr1p: a regulator of cell-surface genes and biofilm formation.</a> Current Biol. 15, 1150-1155 (2005).</li><li>Ramage, G., Saville, S. P., Thomas, D. P., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+biofilms:+an+update.">Candida biofilms: an update.</a> Eukaryot Cell. 4, 633-638 (2005).</li><li>Ramage, G., Vande Walle, K., Wickes, B. L., p, B. L., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+method+for+in+vitro+antifungal+susceptibility+testing+of+Candida+albicans+biofilms.">Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms.</a> Antimicrob Agents Chemother. 45, 2475-2479 (2001).</li><li>Bachmann, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antifungal+combinations+against+Candida+albicans+biofilms+in+vitro.">Antifungal combinations against Candida albicans biofilms in vitro.</a> Antimicrob Agents Chemother. 47, 3657-3659 (2003).</li><li>Ramage, G., VandeWalle, K., Bachmann, S. P., Wickes, B. L., Lopez-Ribot, J. L. <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+pharmacodynamic+properties+of+three+antifungal+agents+against+preformed+Candida+albicans+biofilms+determined+by+time-kill+studies.">In vitro pharmacodynamic properties of three antifungal agents against preformed Candida albicans biofilms determined by time-kill studies.</a> Antimicrob Agents Chemother. 46, 3634-3636 (2002).</li><li>Ramage, G., VandeWalle, K., Wickes, B. L., L&#243;pez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+biofilm+formation+by+Candida+albicans.">Characteristics of biofilm formation by Candida albicans.</a> Rev. Iberoam. Micol. 18, 163-170 (2001).</li><li>Tellier, R., Krajden, M., Grigoriew, G. 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JLL-R posee la equidad en las tecnologías de MicrobeHTS, Inc., que es el desarrollo de agentes antifúngicos. Tecnologías MicrobeHTS, Inc. no proporcionó apoyo financiero para estos estudios.

A continuación se describe una forma simple, rápida, económica y altamente reproducible 96 modelo y placa de microtitulación para la formación de biopelículas de Candida, junto con un método colorimétrico que mide la actividad metabólica de las células dentro del biofilm con XTT. Este modelo 96 y placas de microtitulación para la formación de biofilm fue desarrollado originalmente para C. albicans, pero se puede utilizar para otras especies de Candida. y fácilmente adaptable a otr...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Sabouraud-dextrose agar</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>211584</td>
<td>To prepare plates for fresh subcultures of fungal isolates</td>
</tr>
<tr>
<td>YPD: Yeast peptone dextrose</td>
<td>&nbsp;</td>
<td>US Biological </td>
<td>Y2076</td>
<td>Medium for propagation of overnight liquid cultures</td>
</tr>
<tr>
<td>RPMI-1640 without sodium bicarbonate supplemented with L-glutamine</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>50-020-PB</td>
<td>Liquid medium for biofilm formation</td>
</tr>
<tr>
<td>Morpholinepropanesulfonic acid (MOPS)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP308</td>
<td>To buffer RPMI 1640</td>
</tr>
<tr>
<td>Phosphate buffered saline, PBS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P4417</td>
<td>Buffer for washes</td>
</tr>
<tr>
<td>XTT sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>X4251</td>
<td>See above for preparation instructions</td>
</tr>
<tr>
<td>Ringer's lactate</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC0409-7953-09</td>
<td>For preparation of XTT solution</td>
</tr>
<tr>
<td>Menadione</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5625</td>
<td>Caution: hazardous by skin contact, inhalation or ingestion</td>
</tr>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>15 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430790</td>
<td>&nbsp;</td>
<tr>
<td>50 ml conical centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>96 well microtiter plates: Polystyrene, flat-bottomed, tissue culture treated</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3595</td>
<td>&nbsp;</td>
<tr>
<td>Multichannel pipette and tips</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Incubator</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microtiter plate reader</td>
<td>&nbsp;</td>
<td>Any</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a 96 well microtiter plate-based method for monitoring formation and antifungal susceptibility testing of candida albicans biofilms

Candida albicans es la causa más frecuente de las infecciones por hongos en una creciente población de pacientes inmunocomprometidos y la candidiasis es ahora la tercera enfermedad más común en los hospitales de los EE.UU.. Diferentes manifestaciones de la candidiasis se asocia con la formación de biofilm, tanto en los tejidos del huésped y / o dispositivos médicos (catéteres es decir). Formación de biopelículas lleva consecuencias negativas en la clínica, como las células dentro de los biofilms están protegidos de las respuestas inmunitarias del huésped y de la acción de los antifúngicos. Hemos desarrollado un modelo simple, rápido y robusto in vitro para la formación de C. biofilms albicans de 96 y placas microtiter, que también puede ser utilizado para las pruebas de bio-película de sensibilidad antifúngica. La lectura de este ensayo es colorimétrico, basado en la reducción de XTT (una sal de tetrazolio) por las células metabólicamente activas biopelículas fúngicas. Un experimento típico dura aproximadamente 24 horas para la formación de biofilm, con un adicional de 24 horas para las pruebas de sensibilidad antifúngica. Debido a su simplicidad y el uso de materiales de laboratorio comúnmente disponibles y el equipo, esta técnica democratiza la investigación del biofilm y representa un paso importante hacia la normalización de las pruebas de sensibilidad antifúngica de las biopelículas fúngicas.

Watch this Scientific Journal Video about A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms at JoVE.com

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Se describe un método simple, rápido y robusto para la formación de<em&gt; Candida albicans</em&gt; Biofilms de 96 placas de microtitulación bien y su utilidad en las pruebas de sensibilidad antifúngica de las células dentro de los biofilms.

A 96 de microtitulación y placa basada en el método para la formación de Control y pruebas de sensibilidad antifúngica Candida albicans Las biopelículas

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

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Immunology and Infection

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

44.5K vistas.  University of Texas San Antonio - UTSA. Se describe un método simple, rápido y robusto para la formación de Candida albicans Biofilms de 96 placas de microtitulación bien y su utilidad en las pruebas de sensibilidad antifúngica de las células dentro de los biofilms.

Video: A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Plato de microtitulación de formación de biopelículas de ensayo

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Investigación

Inmunología e Infección

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

En la formación de biofilm in vitro en una diapositiva Cámara de 8 pocillos

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Pruebas de susceptibilidad antimicrobiana de Mycobacterium tuberculosis complex para drogas de primera línea y la segunda por dilución en caldo en un formato de placa de microtitulación

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

Preparación de una Cultura de pellets de sangre para Rapid bacteriana Identificación y Prueba de Susceptibilidad a los antibióticos

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, which are expressed as minimum inhibitory concentrations (MICs) are used in research for antimicrobial development and monitoring of resistance development and in the clinical setting for antimicrobial therapy guidance. Dalbavancin is a semi-synthetic lipoglycopeptide antimicrobial agent that was approved in May 2014 by the Food and Drug Administration (FDA) for the treatment of acute bacterial skin and skin structure infections caused by Gram-positive organisms. The advantage of dalbavancin over current anti-staphylococcal therapies is its long half-life, which allows for once-weekly dosing. Dalbavancin has activity against Staphylococcus aureus (including both methicillin-susceptible S. aureus [MSSA] and methicillin-resistant S. aureus [MRSA]), coagulase-negative staphylococci, Streptococcus pneumoniae, Streptococcus anginosus group, &#946;-hemolytic streptococci and vancomycin susceptible enterococci. Similar to other recent lipoglycopeptide agents, optimization of CLSI and ISO broth susceptibility test methods includes the use of dimethyl sulfoxide (DMSO) as a solvent when preparing stock solutions and polysorbate 80 (P80) to alleviate adherence of the agent to plastic. Prior to the clinical studies and during the initial development of dalbavancin, susceptibility studies were not performed with the use of P-80 and MIC results tended to be 2-4 fold higher and similarly higher MIC results were obtained with the agar dilution susceptibility method. Dalbavancin was first included in CLSI broth microdilution methodology tables in 2005 and amended in 2006 to clarify use of DMSO and P-80. The broth microdilution (BMD) procedure shown here is specific to dalbavancin and is in accordance with the CLSI and ISO methods, with step-by-step detail and focus on the critical steps added for clarity.

A Reference Broth Microdilution Method for Dalbavancin In Vitro Susceptibility Testing of Bacteria that Grow Aerobically

Here, we present a protocol for determination of dalbavancin susceptibility of clinically relevant Gram-positive bacteria using a broth microdilution reference methodology.

Una referencia Caldo método de microdilución para Dalbavancina<em&gt; In Vitro</em&gt; Pruebas de Susceptibilidad de bacterias que crecen en aerobiosis

Antimicrobial susceptibility testing (AST) is performed to assess the in vitro activity of antimicrobial agents against various bacteria. The AST results, ...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Visualización de la formación de Biofilm de Candida albicans utilizando un dispositivo microfluídico automatizado

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Profundo dérmico inyección como un modelo de infección de la piel Candida albicans para el análisis histológico

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic growth of C. albicans SN425, the inoculums grew on Yeast Nitrogen Base media. For biofilm formation, RPMI 1640 media, which have high concentrations of amino acids, were applied to help biofilm growth. Biofilms of 48 h were treated twice a day for a period of 1 min with a non-coherent light device (red light; wavelength = 635 nm; energy density = 87.6 J&#183;cm-2). As a positive control (PC), 0.12% chlorhexidine (CHX) was applied, and as a negative control (NC), 0.89% NaCl was applied to the biofilms. Colony forming units (CFU), dry-weight, soluble and insoluble exopolysaccharides were quantified after treatments. Briefly, the protocol presented here is simple, reproducible and provides answers regarding viability, dry-weight and extracellular polysaccharide amounts after red light treatment.

Daily Phototherapy with Red Light to Regulate Candida albicans Biofilm Growth

Here, we present a protocol to assess the outcome of red light application on the growth of Candida albicans biofilm. A non-coherent red light device with the wavelength of 635 nm and energy density of 87.6 J&#183;cm-2 was applied throughout the growth of Candida albicans biofilms for 48 h.

Diario fototerapia con luz roja para regular crecimiento de Biofilm de Candida albicans

Here, we present a protocol to assess the outcomes of per diem red light treatment on the growth of Candida albicans biofilm. To increase the planktonic ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

Aclaración e imagen de Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Terapia fotodinámica mediada por rosa de Bengala para inhibir Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...