Los autores agradecen el apoyo Mattias Gyllborg instalación calamares y los comentarios sobre este manuscrito, Michael Hadfield y el Laboratorio Marino Kewalo para la asistencia durante la recolección de campo, y los miembros del Laboratorio de Ruby y McFall Ngai-por sus contribuciones a este protocolo. Trabajo en el Laboratorio de Mandel es apoyado por la NSF IOS-0843633.

1. Preparación de inóculos bacterianos <ol><li> Día 0 Dos días antes de la inoculación de calamar, la placa de las cepas bacterianas pertinentes sobre LBS 13 de agar. </li><li> Incubar las bacterias a 25-28 ° C durante la noche. </li><li> Día 1 Inocular 3 ml de medio de LBS en un tubo de cultivo de vidrio con una colonia de cada uno de V. fischeri cepa de infección. Preparar los tubos duplicados como copia de seguridad. </li><li> Día 2 (Coordinar medidas bacterianas 1.4-1.6 con los pasos de calamar 3.7-3.10) 1 h antes de la inoculación, las bacterias subcultivo 1:80 (37,5 l) en 3 ml FTCM en un tubo de cultivo de vidrio y crecer durante 1 hora con aireación. </li><li> Medir el diámetro exterior 600 de la muestra antes de la inoculación. Las mediciones típicas son 0.3-0.6 dependiendo de la cepa. </li><li> Para un inóculo de destino de 3-5 x 10 3 UFC / ml de calcular el volumen del inóculo de la siguiente manera: El volumen de inóculo (l) = 1,25 / OD 600 (por ejemplo, para OD 600 = 0,5, el volumen del inóculo calculado = 1.25/0.5 = 2,5 l). Esta cantidad se añade directamente al agua de mar que contienen calamar en el Paso 4,1. Este cálculo puede ser necesario ajustar para diferentes cepas de V. fischeri o para la inoculación en los niveles inferiores o superiores a los especificados aquí. </li></ol> 2. Preparación de placas de agar para el recuento de los inóculos <ol><li> Para cada tratamiento, LBS etiqueta placas (2 por tratamiento) a las muestras de la placa del inóculo en el Paso 4,1. </li><li> Agregar 5 gotas de enchapado estériles por placa. </li></ol> 3. Colección de los menores de calamar <ol><li> Medir la salinidad del Océano instantánea utilizando el refractómetro y ajustar a 35 ‰. </li><li> Filtro 1 L de Instant Ocean utilizando la unidad de filtración y una línea de vacío adjunto o bomba de vacío, para generar esterilizada por filtración, Instant Ocean (FSIO). Oxigenar el agua por agitación vigorosamente antes de cada dispensación. The la unidad de filtro se puede reutilizar durante 2 días. </li><li> Alícuota 40-50 ml de FSIO en cada uno de dos (2) cuencos de muestra desechables. Etiqueta de uno como Earlies y uno como timelies. </li><li> Preparar un exceso de pipetas de plástico para la adquisición de los calamares juveniles mediante la reducción de la pipeta aproximadamente 1 cm de la punta, por encima de las crestas más bajas (ver Figura 3). Esto facilita un área más amplia a través del cual el calamar puede pasar en el ordeño. Desechar cualquier pipetas de transferencia en los que existe una superficie rugosa expuesta. </li><li> Utilizando pipetas de transferencia preparados, recoger E. scolopes que eclosionaron durante la noche y transferir de un tazón de earlies FSIO. Crías iniciales han sido en el sistema de huevo durante más de 1 h, y son susceptibles a la colonización por contaminar V. fischeri en el sistema de huevo. No utilice earlies para los experimentos de colonización sensibles. </li><li> Comprobar que los tanques de huevo cada minuto 30-45 para las crías nuevas. Asegúrese de que todas las crías se borran en cada cdiablos. Retire las crías con una pipeta de transferencia, y el depósito en el recipiente de timelies FSIO. Los animales recogidos en el momento oportuno se encuentran disponibles para los experimentos de colonización. </li><li> Cuando la colección se ha terminado (~ 45 minutos después del atardecer), la transferencia del calamar al laboratorio principal. Empíricamente, es ventajoso para colonizar los animales bajo condiciones de laboratorio ininterrumpidas de luz para inoculaciones 3 h. </li><li> Para cada tratamiento, preparar un recipiente con 40 ml FSIO. Añadir los calamares a los tazones de fuente para el ensayo (máximo n = 40 por plato). </li><li> Preparar un recipiente adicional como aposymbiotic control (negativo). </li><li> Preparar una pipeta de transferencia específico para cada tratamiento. </li><li> La eutanasia calamares extra en 2% de etanol. </li></ol> 4. Squid Colonización <ol><li> Día 2 - El uso de un Pipetman P10, prescindir de la alícuota calculada de las bacterias (Paso 1.5) en cada tazón calamar (Paso 3.8) para cada tratamiento. Iniciar un temporizador de 3 h inmediatamente después dela primera inoculación. </li><li> Para cada tratamiento, crear un &quot;vórtice&quot; en el recipiente con la pipeta de transferencia dedicado mediante la colocación de la pipeta cerca del borde de la taza y pipeteando arriba y abajo varias veces para mezclar el agua y el calamar durante aproximadamente 10 segundos. La mezcla completa es crítico. </li><li> Placa de 50 l de cada plato en una placa de agar LBS desde el Paso 2.2 (para técnicos repeticiones, dos placas de 50 placas de l por tratamiento). Incubar a 25-28 ° C durante la noche. </li><li> Preparar cuencos de lavado (100 ml FSIO / EA) para cada tratamiento. </li><li> Preparar los viales de Drosophila (4 ml FSIO / EA) para cada calamar. </li><li> Después de exactamente 3 horas, traslado al calamar a sus respectivos lavabos (completo para todo el tratamiento). Esto evita que la inoculación. </li><li> Proceda a la transferencia de cada calamar individuo a su propio vial de Drosophila con FSIO. Utilizar una pipeta de transferencia designada para cada tratamiento. </li><li> Mueva bandejas de frascos de Drosophila a la facilidad de calamar para volver a la día / nDERECHO ciclo de luz de los animales experimentaron durante la embriogénesis. </li><li> Día 3 - Preparar los viales de Drosophila (4 ml FSIO / EA) para cada calamar. </li><li> Antes de atardecer a 22-24 h después de la inoculación, transferir cada calamar de un nuevo vial de Drosophila. Utilizar una pipeta de transferencia designada para cada tratamiento. </li><li> Día 4 - Preparar la etiqueta tubos de microcentrífuga de 1,5 ml (1/squid). </li><li> Antes de atardecer en 46-48 h después de la inoculación, medir y registrar la luminiscencia de cada calamar en el vial de Drosophila (conjunto luminómetro para la integración 6 s, y de auto-lectura sobre el cierre de la tapa). </li><li> Como control negativo para la luminiscencia de fondo, medir un vial con FSIO que no contiene ningún calamar. </li><li> Transferir cada calamar en un volumen de aproximadamente 700 l de un tubo de microcentrífuga de 1,5 ml de la Etapa 4,12. Mover a una caja de cartón del congelador. Una vez que la tapa se coloca en la caja, no lo saque como las señales de luz para la expulsión bacterias no son well-entendido. </li><li> Congelar tubos de microcentrífuga a -80 ° C durante la noche. </li></ol> 5. Determinación de los niveles de colonización <ol><li> Para cada calamar, preparar dos (2) tubos de microcentrífuga, cada uno con 475 l FSIO (o autoclave 70% Instant Ocean). </li><li> Preparar morteros, en primer lugar con un Kimwipe para limpiar la mano del mortero y eliminar los desechos brutos y / o tejidos. </li><li> Colocar morteros punta hacia abajo en un vaso de precipitados de 50 ml que contiene 95% de etanol. El etanol se añade a una altura de aproximadamente 3 cm. </li><li> Por cada mano de mortero, retire del vaso y limpiar la punta con un Kimwipe. </li><li> Sumerja la mano del mortero de nuevo en el baño de etanol, extraer e insertar (punta hacia arriba) en un bastidor de tubo de microcentrífuga y deje que se seque completamente durante aproximadamente 15 minutos. </li><li> Descongele los calamares en un estante de tubo de microcentrífuga (máximo = n = 8). </li><li> Si es necesario, ajustar el volumen a 700 l. </li><li> El uso de un mortero desde el Paso 5.5, alterar el tejido de los animales hasta que el saco de la tinta rupturaras (el agua se torne de un color gris oscuro). </li><li> Retire la mano del mortero y asegurar todo el tejido queda en el tubo. </li><li> Vortex brevemente el tejido durante exactamente 10 segundos (usar un cronómetro). </li><li> Permitir el tejido para descansar durante 10 minutos. El tejido se asienta y las bacterias y la tinta permanece en solución. Para los cálculos que siguen, las bacterias o solución de tinta es la [A] de dilución (es decir, la luz de E. scolopes homogeneizado de órganos en 700 l). Diluciones seriadas 1:20 ([B], [C]) se describen a continuación. </li><li> Para el [B] dilución, se añaden 25 l [A] para uno de los tubos de microcentrífuga preparados en el paso 5,1. Vortex. </li><li> Para la [C] dilución, se añaden 25 l [B] para uno de los tubos de microcentrífuga preparados en el paso 5,1. Vortex. </li><li> Placa de 50 l de cada dilución en agar LBS, 2 repeticiones por tratamiento. </li><li> Incubar las placas a 25-28 ° C durante la noche. </li></ol> 6. Análisis de Datos <ol><li> Para calcular el UFC / órgano de luz (LO), los paísest colonias en la placa para cada tratamiento en el cual 10-400 colonias están presentes, y utilizar la fórmula adecuada: UFC / LO = (colonias en [A] Placa) x 14, o UFC / LO = (colonias en [B] placa) x 280, o UFC / LO = (colonias en [C] placa) x 5600. </li><li> Trazar puntos de datos individuales y medianas en una escala logarítmica. </li><li> Los datos a menudo no se distribuyen normalmente, con diferentes variaciones y los valores extremos puede contener biológicamente significativa de la información. Por lo tanto, no paramétricos pruebas proporcionan un método útil para determinar si los tratamientos difieren significativamente. </li><li> Utilice el software GraphPad Prism para el análisis estadístico. Para ambos tratamientos, el uso del test de Wilcoxon. Para las comparaciones entre más de dos tratamientos, el uso de la prueba de Kruskal-Wallis con el post-tests apropiados. </li></ol> 7. Los resultados representativos Los resultados de un ensayo de colonización de la muestra se muestran en la Figura 4. Dos cepas de <em&gt; V. fischeri que presentan diferentes niveles relativos de la luminiscencia se inocularon en seis calamares, junto con seis de calamar que sirvió como aposymbiotic (colonizada) controles. La E. scolopes simbionte, ES114 14, y la más brillante Sepiola robusta simbionte, SR5 15,16. Niveles similares de inóculo (Fig. 4A) dar lugar a la colonización del 100% a menos de 3 h. A las 48 h, los niveles de luminiscencia (Fig. 4B) y los recuentos de CFU (Fig. 4C) se determinaron para evaluar la aptitud colonización de la cepa. Determinación de la luminiscencia específica (Fig. 4D; bacteria por) permite la determinación del brillo de cada cepa bacteriana durante la simbiosis. <img alt="Figura 1" src="/files/ftp_upload/3758/3758fig1.jpg" /> Figura 1. Diagrama de flujo del procedimiento de colonización. Las bacterias y los calamares se recogen por separado, luego se mezcla en el inóculo se especifica. Calamar se lavan, se transfiere entonces al agua nueva a las 3 h, 24 h, y 4<html> 8 horas después de la inoculación. A las 48 h, la luminiscencia se midió y los animales se congelan, que sirve para esterilizar la superficie de los animales. Luz de órganos colonizados bacterias permanecen viables a -80 ° C a través de un deshielo (no se necesita ciclos hielo-deshielo). <img alt="Figura 2" src="/files/ftp_upload/3758/3758fig2.jpg" /> Figura 2. Diagrama de flujo que ilustra la homogeneización y dilución en placas de las bacterias. Serie de 20 diluciones proporcionar la gama dinámica apropiada para la enumeración de las bacterias colonizadas. <img alt="Figura 3" src="/files/ftp_upload/3758/3758fig3.jpg" /> Figura 3. Transferencia pipetas con una conicidad eje apropiadamente a un agujero estrecho estrecho (A) que dañarían calamar menores. El tratamiento previo al cortar la sección más estrecha con unas tijeras o una hoja de afeitar se obtiene una herramienta apropiada (B) para la transferencia de los calamares juveniles. s/ftp_upload/3758/3758fig4.jpg &quot;/&gt; Figura 4. Datos de ejemplo para un ensayo de colonización. (A) Los niveles de las bacterias en los tazones de fuente de inóculo. (B) La luminiscencia de los calamares individual. (C) recuentos de colonias de calamares individual. (D) luminosidad específica de calamar individual. Apo, Aposymbiotic (colonizada control negativo).

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Los autores no tienen nada que revelar.

El ensayo se describe la colonización permite el análisis de un proceso de simbiosis natural en un entorno controlado de laboratorio. Como tal, se puede utilizar para evaluar la colonización por cepas mutantes, por parte de diferentes aislamientos naturales, y bajo regímenes químicas diferentes. Las variaciones en los experimentos descritos se utilizan comúnmente para evaluar diferentes aspectos de la simbiosis. La cinética de colonización puede ser medida mediante el examen de luminiscencia durante las primeras 24 horas, que puede ser detectada de forma automática en un contador de centelleo en la que se ha eliminado el detector de coincidencia. Además, la capacidad de colonización relativa de una cepa con relación a otro puede ser medida por un ensayo de colonización competitivo, en el que la relación de salida de las dos cepas en un conjunto de animales se normalizan a la relación de entrada (detección diferencial por resistencia a los antibióticos distinta, la fluorescencia , o cromogénicos [LacZ / Xgal Los marcadores). Por último, la colonización puede ser captado directamente por micr confocaloscopy. Es muy importante utilizar los calamares juveniles saludable para los experimentos. Los indicadores del comportamiento de la mala salud incluyen calamares nadando en círculos (la eutanasia del animal), o de los animales que permanecen blancas y no alteran sus cromatóforos a tomar color sobre una superficie oscura (usar con cuidado). Como no existe variación en las poblaciones de acogida, un mayor número de replicar los experimentos con menor número de animales es a menudo más valioso que un menor número de replicar los experimentos con muestras de gran tamaño.

<table border="1"><tbody><tr><td> Nombre de reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Tubos de vidrio Cultura, 16 mm de diámetro </td><td> VWR </td><td> 47729-580 </td><td></td></tr><tr><td> Tapas para tubos de cultivo de vidrio </td><td> Pescador </td><td> NC9807998 </td><td></td></tr><tr><td> Espectrofotómetro visible para la determinación de OD 600 </td><td> Biowave </td><td> CO8000 </td><td> Cualquier espectrofotómetro capaz de medir DO 600 va a funcionar. Esta unidad puede medir el diámetro exterior 600 de líquido directamente en los tubos de vidrio de cultivo. Algunos de ajuste del cálculo de inóculo puede ser necesario dependiendo del instrumento utilizado. </td></tr><tr><td> GloMax 20/20 de un solo tubo luminómetro </td><td> Promega </td><td> E5311 </td><td> Equivalente a la Turner Biosystems 20/20n luminómetro. Incluye el soporte del tubo de microcentrífuga. </td></tr><tr><td> GloMax 20/20 Norma Luz </td><td> Promega </td><td> E5341 </td><td> Para la calibración del luminómetro. </td></tr><tr><td> Refractómetro de mano </td><td> Foster y Smith Aquatics </td><td> CD-14035 </td><td> Calibrar antes de cada uso con agua desionizada. Enjuagar después de cada uso con agua desionizada para evitar la acumulación de sal. </td></tr><tr><td> Instant Ocean (concentrado de agua de mar artificial) </td><td> Foster &amp; Smith Aquatics </td><td> CD-16881 </td><td> Preparar a 35 ‰ en agua desionizada, utilizando el refractómetro, a continuación, a través de un filtro de 0,2 micras SFCA filtro. </td></tr><tr><td> Unidad de filtración </td><td> Nalgene </td><td> 158-0020 </td><td> El surfactante libre de acetato de celulosa (SFCA) de la membrana, de 0,2 m. Hemos observado resultados variables, con algunos filtros que contienen surfactantes de PSA. </td></tr><tr><td> Pipetas de transferencia </td><td> Pescador </td><td> 13-711 -9 a.m. </td><td> Con una tijera o cuchilla de afeitar, cortar la punta limpiamente por encima de la cresta primero para aumentar el diámetro de la punta de la pipeta y evitar apretar las crías de calamar. </td></tr><tr><td> Tazones de muestra desechables (vasos de plástico) </td><td> Cometa </td><td> T9S (9 oz.) </td><td> Tazones para la inoculación, con diámetro superior de 3 ¼ &quot;, de diámetro menor de 2 ¼&quot;, altura de 3 &quot;. Bowls crear un entorno homogéneo, ya que no tienen borde inferior, en la que el calamar puede quedar atrapado en un nicho de bajo oxígeno. El tamaño se ha optimizado de 40 ml de inóculo. Disponible en webstaurantstore.com, # 619PI9. </td></tr><tr><td> Drosophila Viales </td><td> VWR </td><td> 89092-720 </td><td> De diámetro Vial coincide con la apertura en el PMT luminómetro. </td></tr><tr><td> Tubos de microcentrífuga de 1,5 ml </td><td> ISC Bioexpress </td><td> C-3217-1CS </td><td> Los tubos deben adaptarse a la forma de las manos de mortero. </td></tr><tr><td> El etanol, Prueba 200 </td><td> Pescador </td><td> BP2818-100 </td><td></td></tr><tr><td> Pistilos </td><td> Kimble Chase / Kontes </td><td> 749521-1500 </td><td></td></tr><tr><td> Cuentas de enchapado, 5 mm de diámetro </td><td> Kimble Chase, </td><td> 13500 5 </td><td> Preparar 5 por tubo y autoclave. </td></tr></tbody></table>

colonization of euprymna scolopes squid by vibrio fischeri

Bacterias específicas se encuentran en asociación con el tejido animal 1-5. Estas bacterias de acogida las asociaciones (simbiosis) pueden ser perjudiciales (patógeno), no tienen ninguna consecuencia de fitness (comensales), o ser beneficiosa (mutualismo). Si bien se ha prestado mucha atención a las interacciones patógenas, poco se sabe acerca de los procesos que determinan la adquisición reproducible de beneficio / comensal bacterias del medio ambiente. El mutualismo de luz de órganos entre la marina bacteria Gram negativa V. fischeri y el calamar hawaiano bobtail, E. scolopes, representa una interacción muy específico en el que un único equipo (E. scolopes) establece una relación simbiótica con una sola especie bacteriana (V. fischeri) a lo largo de su ciclo de vida 6,7. La bioluminiscencia producida por V. fischeri durante esta interacción proporciona un beneficio anti-depredadores a E. scolopes durante las actividades nocturnas, mientras que 8,9el tejido del huésped, rica en nutrientes proporciona V. fischeri con un nicho protegido 10. En cada generación de acogida, esta relación se resume, por lo que representa un proceso predecible que puede ser evaluado en detalle en las distintas etapas de desarrollo simbiótico. En el laboratorio, el menor calamar escotilla aposymbiotically (uncolonized), y, si se recogen dentro de los primeros 30-60 minutos y se transfiere a simbionte agua libre, no puede ser colonizado excepto por el inóculo experimental 6. Esta interacción proporciona así un sistema modelo útil en la cual evaluar los pasos individuales que conducen a la adquisición específica de un microbio simbiótica desde el entorno de 11,12. Aquí se describe un método para evaluar el grado de colonización que se produce cuando recién nacidas aposymbiotic E. scolopes están expuestos a (artificial) de agua de mar que contienen V. fischeri. Este ensayo simple describe la inoculación, la infección natural, y la recuperaciónde bacterias simbiontes del órgano de luz naciente de la E. scolopes. Hay que tener cuidado para proporcionar un entorno uniforme para los animales durante el desarrollo simbiótico, especialmente con respecto a la calidad del agua y las señales de luz. Métodos para caracterizar la población simbiótica descritos incluyen (1) la medición de bioluminiscencia por bacterias derivados, y (2) directo de las colonias el recuento de los simbiontes recuperados.

Watch this Scientific Journal Video about Colonization of Euprymna scolopes Squid by Vibrio fischeri at JoVE.com

Colonization of Euprymna scolopes Squid by Vibrio fischeri

El método describe el procedimiento mediante el cual el hawaiano calamar bobtail,<em&gt; Euprymna scolopes</em&gt; Y su simbionte bacteriano,<em&gt; Vibrio fischeri</em&gt;, Se elevan por separado y después se introdujo para permitir la colonización específica del órgano de luz calamar por las bacterias. Detección colonización por bacterias luminiscencia-derivado y por recuento directo de las colonias se describen.

La colonización de Euprymna scolopes Squid por Vibrio fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

colonization-of-euprymna-scolopes-squid-by-vibrio-fischeri

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Colonization of Euprymna scolopes Squid by Vibrio fischeri

19.8K vistas.  Northwestern University. El método describe el procedimiento mediante el cual el hawaiano calamar bobtail, Euprymna scolopes Y su simbionte bacteriano, Vibrio fischeri, Se elevan por separado y después se introdujo para permitir la colonización específica del órgano de luz calamar por las bacterias. Detección colonización por bacterias luminiscencia-derivado y por recuento directo de las colonias se describen.

Video: Colonization of Euprymna scolopes Squid by Vibrio fischeri

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

La evaluación de alteraciones hepáticas metabólicas durante la colonización progresiva de la libre de gérmenes de ratón por 1 H espectroscopía de RMN

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Investigación

Inmunología e Infección

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

La cuantificación de la colonización por hongos, esporogenesis y producción de micotoxinas mediante bioensayos del núcleo

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

TransFLP - Un método para modificar genéticamente<em&gt; Vibrio cholerae</em&gt; Sobre la base de la transformación natural y la recombinación FLP

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Caracterización De Las Respuestas Inflamatorias Durante La Colonización Intranasal Con Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

La investigación de los efectos de los probióticos en antineumocócica Colonización El uso de un<em&gt; In Vitro</em&gt; Ensayo de adherencia

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

Evaluación De La Invasión Bacteriana De Las Células Cardíacas En Cultivo Y Colonización Del Corazón En Ratones Infectados Usando Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Desarrollo de una más sensible y específica cromogénico medio de agar para la detección de<em&gt; Vibrio parahaemolyticus</em&gt; Y Otros<em&gt; Vibrio</em&gt; Especies

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonización vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

Técnicas de laboratorio utilizadas para mantener y diferenciar los biotipos de<em&gt; Vibrio cholerae</em&gt; Aislados Clínicos y Ambientales

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

Inducción de la diferenciación celular y de imágenes monocelulares de<em&gt; Vibrio parahaemolyticus</em&gt; Células Swimmer y Swarmer

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...