Los autores desean agradecer a junio Middleton para el suministro de E. cepas faecalis y de orientación con cultivo de bacterias. C. elegans fueron proporcionados por la CGC, que es financiado por el NIH Oficina de Programas de Infraestructura de Investigación (P40 OD010440).

1) Preparación de nematodos Medio de Crecimiento (NGM) <ol><li> Para hacer 50 platos, añadir 1,5 g de NaCl, 8,5 g Agar ultrapura, y 1,25 g de peptona al 1 Lflask. </li><li> Añadir 487,5 ml de H 2 O al matraz. Agitar suavemente para mezclar y cubrir la apertura del frasco con un trozo de papel de aluminio. </li><li> Esterilizar la solución en autoclave a condiciones estándar (121 psi, 120 ° C, 20 min). </li><li> Dejar que la solución se enfríe a 45 ° C en un baño de agua. </li><li> A la solución, agregue el siguiente en orden: 500 l de colesterol a 5 mg / ml solución madre (en etanol), 500 l 1 M CaCl2, 500 l 1 M MgSO4, y 12,5 ml KPO4 tampón (54,15 g KH 2 PO 4 y 17,8 g K 2 HPO 4 en 500 ml de H 2 O), como se describe en el 18. </li><li> Uso de pipetas estériles, añadir 10 ml de solución de agar a cada 60 mm de placa de Petri de poliestireno. Permita que el medio a solidificarse a temperatura ambiente Overnight. </li></ol> 2) Preparación de B caldo de E. coli Medios <ol><li> Para hacer cinco 100 ml de cultivos, añadir 5,0 g de Bacto triptona, 2,5 g de extracto de levadura, 5,0 g de NaCl a un vaso de precipitados. </li><li> Añadir agua destilada estéril hasta un volumen final de 500 ml. Calentar la solución en una placa caliente, mientras se agita, hasta que los solutos se han disuelto. </li><li> Verter 100 ml de caldo de B en cinco botellas de vidrio (por lo menos 200 ml de volumen). </li><li> Esterilizar en autoclave las soluciones en condiciones normales (121 psi, 120 ° C, 20 minutos). </li><li> Permitir caldo B se enfríe a temperatura ambiente antes de su uso. </li></ol> 3) Siembra C. Placas Mantenimiento elegans <ol><li> Después de permitir que NGM placas se sequen a temperatura ambiente durante al menos una semana, uno inocular 100 ml de caldo de cultivo B con unas pocas colonias de E. coli (OP50). </li><li> Crecer E. cultura coli a 37 ° C durante la noche. </li><li> Pipeta una o dos gotas (aproximadamente 10081; l) de OP50 cultura en el centro de cada placa de NGM. Las placas se apilan normalmente tapa hacia arriba. Cuando pipeteado (siembra), comenzar con la pipeta la cultura sobre la placa en la parte inferior de la pila. Su forma de trabajo de cada pila. Trate de no mover las placas después de la siembra, ya que es importante que la cultura no se acerque demasiado a los bordes de la placa. </li><li> Permitir a las placas sembradas se sequen a temperatura ambiente durante al menos una semana antes de usar. </li><li> Placas sembradas se almacenan (invertida) en recipientes de plástico sellados a temperatura ambiente. </li></ol> 4) Mantenimiento C. elegans cepas <ol><li> Para mantener bien alimentados C. stocks elegans, un gusano utilizan elegir para pasar tres L4s o adultos grávidas a una nueva, sin semillas NGM placas cada 3-4 días. </li><li> Placas Tienda (invertida) preferentemente dentro de un incubador a 15 - 22 º C. Culturas en los ensayos descritos fueron almacenadas a 20 ° C. C. elegans desarrollo es dependiente de la temperatura. Como maintenana vez que aumenta la temperatura, el tiempo entre las etapas de desarrollo disminuye. </li></ol> 5) Preparación de agar de tripticasa de soja (TSA) E. faecalis Cultura Medios <ol><li> Para 50 placas, mida 500 ml de agua destilada estéril en un frasco y llevar a ebullición, mientras se agita, en una placa calefactora. </li><li> Añadir 20 g de agar TSA en polvo a un matraz y permita que la solución para disolver. </li><li> Esterilizar en autoclave las soluciones en condiciones normales (121 psi, 120 ° C, 20 minutos). </li><li> Dejar que la solución se enfríe a 45 ° C en un baño de agua. </li><li> Uso de pipetas estériles, añadir 10 ml de solución de agar a cada 60 mm de placa de Petri de poliestireno. Permita que el medio se solidifican a temperatura ambiente durante la noche. </li></ol> 6) Preparación de caldo de soja tripticasa (TSB) E. faecalis Cultura Medios <ol><li> Para hacer 250 tubos de cultivo, mida 500 ml de agua destilada estéril en un frasco y acogedor en una placa calefactora con agitación. </li&gt; <li> Añadir 15 g de polvo de agar TSB al matraz y permitir que el polvo se disuelva. </li><li> Esterilizar en autoclave las soluciones en condiciones normales (121 psi, 120 ° C, 20 minutos). </li><li> Deje que la solución se enfríe a temperatura ambiente, a continuación, pipeta 2 ml en tubos de ensayo, 5 ml estériles. </li></ol> 7) Elaboración de medios de infusión cerebro corazón (BHI), con estreptomicina, por E. faecalis Cultura <ol><li> Medir 500 ml de agua destilada estéril en un frasco y llevar a ebullición, mientras se agita, en una placa calefactora. </li><li> Añadir 26 g de medio BHI en el matraz y permita que la solución para disolver. </li><li> Esterilizar en autoclave las soluciones en condiciones normales (121 psi, 120 ° C, 20 minutos). </li><li> Deje que la solución se enfríe a 45 ° C en un baño de agua. </li><li> Utilizando una técnica estéril, añadir 0,5 ml de una solución madre de estreptomicina y agitar para mezclar 10 mg / ml. </li><li> Añadir 10 ml de la solución a cada uno de 60 mm de placa de Petri de poliestireno. </li><li&gt; Permita que el medio para solidificar a temperatura ambiente durante la noche. Placas Tienda (invertida) a 4 ° C hasta pocas horas antes de su uso. </li></ol> 8) Mantener E. faecalis cepas <ol><li> Para mantener E. stocks faecalis, use un asa estéril de racha colonias individuales en placas de agar de soja tríptico separados (TSA). </li><li> Los cultivos a 37 ° C durante la noche. Placas Tienda (invertida) en una bolsa sellada o caja a temperatura ambiente durante varias semanas. </li></ol> 9) Preparar E. Placas faecalis para EIW Ensayo <ol><li> Inocular 2 ml de un cultivo TSB con una colonia de E. faecalis y se incuba durante la noche a 37 ° C. </li><li> Pipetear 20 l de E. cultura faecalis en el centro de una placa de BHI-estreptomicina preparado, y se incuban durante la noche a 37 ° C. </li></ol> 10) Preparación de E. Placas coli para EIW ensayo (placas de control) <ol><li> Inocular un ml B-bro 100ª cultivo con una colonia de E. OP50 coli y se incuban durante la noche a 37 ° C </li><li> Pipetear 20 l de la cultura OP50 en el centro de una placa de NGM cabeza de serie y permitir que las placas sembradas se sequen a temperatura ambiente durante la noche. </li></ol> 11) Egg en el Ensayo Gusano <ol><li> Elige 15-20 organizado-L4 C. elegans (Figura 1) en el centro de un césped de bacterias en un preparado BHI-Strep-E. placa faecalis. Tenga cuidado de no transferir una gran cantidad de OP50 a la placa BHI. Elija el mismo número de L4s a un control NGM-OP50 plato. </li><li> Incubar las placas durante 40 horas a 20 ° C. </li><li> Prepare una solución de lejía al 20%. Mezclar una lejía comercial (6,0% de hipoclorito de sodio) con el volumen apropiado de agua destilada. </li><li> Añadir 10 l gotas de lejía por diez lugares distintos en una tapa de plástico (de un gusano o placa bacteriana). </li><li> Con una selección de gusano, transferir un gusano en cada gota de lejía. Agite suavemente la punta de larecoger en la gota cloro para asegurarse de que el gusano se ha lavado de la final de la selección (confirmar al ver la gota en un microscopio). </li><li> Permitir la cutícula para disolver durante aproximadamente 10 minutos o hasta que los gusanos se abrieron de golpe, la expulsión de los huevos (Figura 2). Tenga cuidado de no trasladar los huevos a la plancha. </li><li> El uso de un microscopio de disección, contar los huevos en cada gota de lejía. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Los pasos más importantes en la realización con éxito este ensayo son: 1) el uso de acciones bien alimentados de C. elegans, 2) cultivar tipos individuales de bacterias en la placa de ensayo, 3) identificar con precisión por etapas L4 gusanos para la exposición a E. faecalis, 4) mantener el tiempo de exposición a E. faecalis consistentes en todos los juicios y 5) el tiempo de blanqueo no debe exceder de diez minutos para evitar la desintegración de huevo. Pa...

Caenorhabditis elegans, un gusano redondo microscópica, de vida libre, es un organismo modelo utilizado tradicionalmente para estudiar los procesos de desarrollo y la señalización celular, debido a su anatomía transparente, el desarrollo bien caracterizado, totalmente secuenciado del genoma, en tiempo de generación corto, y la homología genética de los seres humanos . Más recientemente, C. elegans se ha convertido en un organismo modelo en el campo de la toxicología del medio ambiente y la inmunidad innata 10, 11. Estos gusanos hermafroditas auto-fertilizantes alcanzan la madurez sexual a los dos o tres días de la eclosión del huevo. Durante su ciclo de vida, C. elegans pasa por cuatro estadios larvales (L1-L4), antes de llegar a la edad adulta. Un aislado hermafrodita puede producir, en promedio, 300 crías dentro de los tres días de la fecundidad alta. En la madurez reproductiva C. elegans hermafroditas, los huevos fertilizados son retenidos dentro del útero durante varias horas antes de ser establecido. Lanúmero normal de los huevos almacenados en el útero a la vez (durante el pico fecundidad) es de entre diez y quince 12. El número de huevos en el útero es una función tanto de la tasa de producción de huevos y la tasa de puesta de huevos. Los huevos fertilizados son expulsados ​​del útero por la contracción de los músculos vulvares dieciséis dispuestas alrededor de la abertura de la vulva 13. Motoneuronas Hermafrodita específicos (HSN &#39;s) y neuronas motoras VC sinapsis en los músculos vulvares que afectan a la contracción muscular y por lo tanto la puesta de huevos comportamiento 5,7,13,14. La expulsión de los huevos desde el útero se produce debido a la actividad coordinada de las neuronas y los músculos. Cultivos de laboratorio de C. elegans se plantean típicamente en una dieta de no patógenas de Escherichia coli OP50. En el medio natural, C. elegans entran en contacto con una variedad de fuentes de alimentos, tales como bacterias patógenas, que pueden ser potencialmente dañinos. Cuando se expone a sustancias nocivas enel medio ambiente, C. elegans retienen los huevos hasta que se convierte en el medio ambiente más favorable. Es de suponer que esta retención de huevo es un esfuerzo para proteger a sus crías. En este huevo en el ensayo, C. gusano (EIW) elegans están expuestos a las bacterias potencialmente patógenas, Enterococcus faecalis, que se encuentra en el medio ambiente. La exposición a formas patógenas de E. faecalis puede causar una infección intestinal persistente e incluso la muerte en C. elegans 15. La exposición a otras formas de bacterias patógenas han sido demostrado que afectan a la retención de huevo 16,17, sin embargo, el efecto no se cuantificó. Además, el efecto de las cepas patógenas de E. ligeramente faecalis, cepas que no son inmediatamente letal, en el comportamiento de puesta de huevos no se ha estudiado. EIW ensayos implican contando el número de huevos retenidos en el útero de C. elegans 4. A pesar de que C. elegansson transparentes, los huevos se acumulan en el útero pueden ser difíciles de cuantificar en un animal intacto. El ensayo consiste en EIW blanqueo grávido adultos C. elegans que fueron expuestos a las bacterias por un período fijo de tiempo. La solución de cloro se disuelve la cutícula exterior dejando detrás de los huevos. Los huevos son de refracción a los efectos de cloro debido a la presencia de una cáscara de huevo. Después del blanqueo, uno es muy fácilmente capaz de contar el número de óvulos liberados desde el útero de los gusanos a blanqueo. El ensayo descrito es un método simple, barato y rápido para cuantificar el número de huevos en el útero de una sola vez, y por lo tanto cuantificar los efectos de E. faecalis sobre la retención de huevos. Este ensayo se puede utilizar para cuantificar el efecto de otros tipos de bacterias, toxinas ambientales o fármacos sobre la retención de huevo. Este ensayo también tiene el potencial para ser utilizado como una pantalla para la patogenicidad bacteriana.

<table><tbody><tr><td>Agar, ultrapure</td><td>Affymetrix</td><td>10906</td><td></td></tr><tr><td>Bacto Peptone</td><td>Becton Dickinson</td><td>211677</td><td></td></tr><tr><td>Bacto Tryptone</td><td>Becton Dickinson</td><td>211705</td><td></td></tr><tr><td>Brain Heart Infusion dehydrated medium</td><td>Carolina Biological Supply</td><td>781781</td><td></td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt;, N2 strain</td><td>Caenorhabditis Genetics Center</td><td></td><td>http://www.cbs.umn.edu/cgc</td></tr><tr><td>Cholesterol</td><td>Alfa Aesar</td><td>A11470</td><td></td></tr><tr><td>Culture plates for &lt;em&gt;C. elegans&lt;/em&gt;</td><td>Tritech Research Inc.</td><td>T3308</td><td></td></tr><tr><td>Culture plates for &lt;em&gt;E. faecalis&lt;/em&gt;</td><td>Fisher Scientific-Fisherbrand</td><td>875713</td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;(OP50)</td><td>Caenorhabditis Genetics Center</td><td></td><td>http://www.cbs.umn.edu/cgc</td></tr><tr><td>&lt;em&gt;E. faecalis&lt;/em&gt; strains</td><td></td><td></td><td>provided by J. Middleton. All isolates were confirmed as enterococci</td></tr><tr><td colspan="4">by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td></tr><tr><td colspan="4"> &lt;p&gt;all strains grew at 44.5 &amp;ordm;C and were catalase negative and hydrolyzed esculin. A simplified&amp;nbsp;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;dichotomous key based on pigmentation and fermentation reactions for six sugars&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;(arabinose, mannitol, methyl-&amp;alpha;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;presumptive identification of all &lt;em&gt;E&lt;/em&gt;. &lt;em&gt;faecalis&lt;/em&gt; strains (Efs lacks pigmentation and is arabinose, MGP&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&amp;nbsp;&lt;/span&gt;&lt;span style=&quot;line-height: 1.6em;&quot;&gt;were confirmed using the API 20 STREP system (Biomerieux).&lt;/span&gt;&lt;/p&gt; </td></tr><tr><td>Microscope</td><td>Motic</td><td>SMZ 168B</td><td>any microscope with transmitted illumination and 50X magnification should be sufficient</td></tr><tr><td>Streptomycin sulfate</td><td>Fisher BioReagents</td><td>BP910-50</td><td></td></tr><tr><td>Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td><td>Becton Dickinson</td><td>236950</td><td></td></tr><tr><td>Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td><td>Becton Dickinson</td><td>211768</td><td></td></tr><tr><td>Yeast extract</td><td>Acros</td><td>61180-1000</td><td></td></tr></tbody></table>

measuring the effects of bacteria on c. elegans behavior using an egg retention assay

C. elegans comportamiento de puesta de huevos se ve afectada por señales ambientales tales como la osmolaridad 1 y la vibración 2. Ante la falta total de alimentos C. elegans también dejan la puesta de huevos y retienen los huevos fertilizados en el útero 3. Sin embargo, el efecto de diferentes fuentes de alimentos, especialmente bacterias patógenas y en particular de Enterococcus faecalis, en el comportamiento de puesta de huevos no está bien caracterizado. El ensayo de huevo-en-gusano (EIW) es una herramienta útil para cuantificar los efectos de diferentes tipos de bacterias, en este caso E. faecalis, en el comportamiento de puesta de huevos. EIW ensayos implican contando el número de huevos retenidos en el útero de C. elegans 4. El ensayo consiste en EIW blanqueo etapas, grávido adultos C. elegans para quitar la cutícula y se separan los huevos retenidos desde el animal. Antes de blanqueo, los gusanos están expuestos a las bacterias (o cualquier tipo de señal ambiental) Durante un período de tiempo fijo. Después del blanqueo, uno es muy fácilmente capaz de contar el número de huevos retenidos en el interior del útero de los gusanos. En este ensayo, un aumento cuantificable en la retención de huevo después de E. la exposición faecalis se puede medir fácilmente. El ensayo EIW es un ensayo de comportamiento que puede ser utilizado para la detección de bacterias potencialmente patógenas o la presencia de toxinas del medio ambiente. Además, el ensayo de EIW puede ser una herramienta para la detección de drogas que afectan neurotransmisor de señalización ya que el comportamiento puesta de huevos es modulada por los neurotransmisores como la serotonina y la acetilcolina 5-9.

Este ensayo permite cuantificar el número de huevos retenidos en C. elegans después de la exposición a E. faecalis. L4 escena gusanos (caracterizado por el espacio abierto transparente por encima de su vulva, Figura 1) fueron expuestos a E. faecalis a través de la edad adulta. Después de cuarenta horas de exposición a E. faecalis, se llevó a cabo el blanqueo. A medida que la cutícula se desintegró en la gota de lejía, los huevos se hicieron más evidentes <s...

Watch this Scientific Journal Video about Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay at JoVE.com

Medición de los efectos de las bacterias sobre el comportamiento de C. Elegans mediante un ensayo de retención de óvulos | Biología | Video

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Un ensayo de huevo-en-gusano (EIW) es un método útil para cuantificar el comportamiento de puesta de huevos. Las alteraciones de la postura de huevos pueden ser una respuesta de comportamiento del organismo modelo<em&gt; Caenorhabditis elegans</em&gt; A sustancias ambientales potencialmente nocivas, tales como las producidas por bacterias patógenas.

Medición de los efectos de las bacterias en<em&gt; C. Elegans</em&gt; Comportamiento El uso de un ensayo de retención de huevo

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

measuring-effects-bacteria-on-c-elegans-behavior-using-an-egg

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Biology

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Ecology

Behavior

SCIENTISTS IN ACTION

17.1K vistas.  Fairleigh Dickinson University. Un ensayo de huevo-en-gusano (EIW) es un método útil para cuantificar el comportamiento de puesta de huevos. Las alteraciones de la postura de huevos pueden ser una respuesta de comportamiento del organismo modelo Caenorhabditis elegans A sustancias ambientales potencialmente nocivas, tales como las producidas por bacterias patógenas.

Video: Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

El nematodo Caenorhabditis Elegans - un modelo versátil en Vivo para el estudio de las interacciones huésped-microbio

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Investigación

Genética

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

Análisis Automatizado de un Ensayo de Preferencia Chemosensorial Basada en Población de Nematodo

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Comportamiento

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

Encuesta de métodos de bajo costo para medir la esperanza de vida y la salud en Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

Un ensayo para medir los efectos del etanol sobre la velocidad de locomoción<em&gt; Caenorhabditis elegans</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Neurociencias

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

UN<em&gt; Caenorhabditis elegans</em&gt; Ensayo de aversión al cobre basado en el estado nutricional

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Medir el efecto de productos químicos sobre el crecimiento y reproducción de Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

Medicina

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans Ensayo de quimiotaxis

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Biología

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Inactivación metabólica bacteriana en Caenorhabditis elegans mediante paraformaldehído

Methodology to Metabolically Inactivate Bacteria for Caenorhabditis elegans Research

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a diet of live bacteria, the metabolic activity of their food source can confound experiments looking for the direct effects of various interventions on the worm. To avoid the confounding effects of bacterial metabolism, C. elegans researchers have used multiple methods to metabolically inactivate bacteria, including ultraviolet (UV)-irradiation, heat-killing, and antibiotics. UV treatment is relatively low-throughput and cannot be used in liquid culture because each plate must be examined for successful bacterial killing. A second treatment method, heat-killing, negatively affects the texture and nutritional quality of the bacteria, leading to the developmental arrest of C. elegans. Finally, antibiotic treatment can directly alter C. elegans physiology in addition to preventing bacterial growth. This manuscript describes an alternative method to metabolically inactivate bacteria using paraformaldehyde (PFA). PFA treatment cross-links proteins within bacterial cells to prevent metabolic activity while preserving cellular structure and nutritional content. This method is high-throughput and can be used in liquid culture or solid plates, as testing one plate of PFA-treated bacteria for growth validates the whole batch. Metabolic inactivation through PFA treatment can be used to eliminate the confounding effects of bacterial metabolism on studies of drug or metabolite supplementation, stress resistance, metabolomics, and behavior in C. elegans.

Autor destacado: Avance de la investigación de Caenorhabditis elegans utilizando bacterias tratadas con paraformaldehído 

The food source for Caenorhabditis elegans in the lab is live Escherichia coli. Since bacteria are metabolically active, they present a confounding variable in metabolic and drug studies in C. elegans. A detailed protocol to metabolically inactivate bacteria using paraformaldehyde is described here.

Metodología para la inactivación metabólica de bacterias para la investigación de Caenorhabditis elegans

Caenorhabditis elegans is a common model organism for research in genetics, development, aging, metabolism, and behavior. Because C. elegans consume a ...

Medición de los efectos de las bacterias en

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