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 border="0" cellpadding="0" cellspacing="0" width="2472">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Agar, ultrapure</td>
			<td style="width:191px;">Affymetrix</td>
			<td style="width:119px;">10906</td>
			<td style="width:1791px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Peptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Bacto Tryptone</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211705</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Brain Heart Infusion dehydrated medium</td>
			<td style="width:191px;">Carolina Biological Supply</td>
			<td style="width:119px;">781781</td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">C. elegans, N2 strain</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:371px;">Cholesterol</td>
			<td style="width:191px;">Alfa Aesar</td>
			<td style="width:119px;">A11470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Culture plates for C. elegans</td>
			<td style="width:191px;">Tritech Research Inc.</td>
			<td style="width:119px;">T3308</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Culture plates for E. faecalis</td>
			<td style="width:191px;">Fisher Scientific-Fisherbrand</td>
			<td style="width:119px;">875713</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">E. coli (OP50)</td>
			<td style="width:191px;">Caenorhabditis Genetics Center</td>
			<td style="width:119px;"></td>
			<td>http://www.cbs.umn.edu/cgc</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">E. faecalis strains</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>provided by J. Middleton. All isolates were confirmed as enterococci</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>by observing growth on enterococcosel agar (BBL) and in 6% NaCl broth;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td>
			all strains grew at 44.5 &#186;C and were catalase negative and hydrolyzed esculin. A simplified&#160;dichotomous key based on pigmentation and fermentation reactions for six sugars&#160;(arabinose, mannitol, methyl-&#945;-D-glucopyranoside (MGP), ribose, sorbose and sorbitol) allowed&#160;presumptive identification of all E. faecalis strains (Efs lacks pigmentation and is arabinose, MGP&#160;and sorbose negative and sorbitol, mannitol and ribose positive). All presumptive Efs strains&#160;were confirmed using the API 20 STREP system (Biomerieux).
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;"></td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microscope</td>
			<td style="width:191px;">Motic</td>
			<td style="width:119px;">SMZ 168B</td>
			<td>any microscope with transmitted illumination and 50X magnification should be sufficient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Streptomycin sulfate</td>
			<td style="width:191px;">Fisher BioReagents</td>
			<td style="width:119px;">BP910-50</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Tryptic Soy Agar (Soybean-Casein Digest Agar Medium), Difco</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">236950</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:371px;">Trypticase Soy Broth (Soybean-Casein Digest Medium), BBL</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:119px;">211768</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Yeast extract</td>
			<td style="width:191px;">Acros</td>
			<td style="width:119px;">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

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

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

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16.9K 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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Generación de transgénicos estable C. elegans con microinyección

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Investigación

Biología

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Medición Caenorhabditis elegans Duración de la vida en el medio sólido

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imágenes C. elegans Los embriones utilizando un microscopio de epifluorescencia y software de código abierto

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

La visualización de la función respiratoria mitocondrial utilizando citocromo C Oxidasa / Succinato Deshidrogenasa (COX / SDH) de doble etiquetado Histoquímica

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Creación de ambientes definidos de gases para el Estudio de los efectos de la hipoxia en el<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Un ensayo de explante para evaluar el comportamiento celular del mesénquima craneal

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

Tinción de anticuerpos en<em&gt; C. Elegans</em&gt; Uso de &quot;Freeze-Cracking&quot;

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well when a small number of genes are targeted for knockdown, large scale feeding screens show variable knockdown efficiencies, which limits their utility. We have deconstructed previously published RNAi knockdown protocols and found that the primary source of the reduced knockdown can be attributed to the loss of dsRNA-encoding plasmids from the bacteria fed to the animals. Based on these observations, we have developed a dsRNA feeding protocol that greatly reduces or eliminates plasmid loss to achieve efficient, high throughput knockdown. We demonstrate that this protocol will produce robust, reproducible knock down of C. elegans genes in multiple tissue types, including neurons, and will permit efficient knockdown in large scale screens. This protocol uses a commercially available dsRNA feeding library and describes all steps needed to duplicate the library and perform dsRNA screens. The protocol does not require the use of any sophisticated equipment, and can therefore be performed by any C. elegans lab.

Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

While dsRNA feeding in C. elegans is a powerful tool to assess gene function, current protocols for large scale feeding screens result in variable knockdown efficiencies. We describe an improved protocol for performing large scale RNAi feeding screens that results in highly efficacious and reproducible knockdown of gene expression.

Derribo Gene a gran escala en<em&gt; C. elegans</em&gt; Utilizar bibliotecas de alimentación de dsRNA para generar sólidas fenotipos de pérdida de función

RNA interference by feeding worms bacteria expressing dsRNAs has been a useful tool to assess gene function in C. elegans. While this strategy works well ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Un Modelo Anoxia inanición por isquemia / reperfusión en el<em&gt; C. elegans</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...