Les auteurs tiennent à remercier Juin Middleton pour fournir E. souches faecalis et d&#39;orientation à la culture des bactéries. C. elegans ont été fournis par la CCG, qui est financé par le NIH Bureau des programmes d&#39;infrastructure de recherche (P40 OD010440).

1) Préparation de nématodes croissance médias (NGM) <ol><li> Pour faire 50 assiettes, ajouter 1,5 g NaCl, 8,5 g Ultrapure Agar, et 1,25 g de peptone à 1 Lflask. </li><li> Ajouter 487,5 ml de dH 2 O dans le ballon. Agiter doucement pour mélanger et couvrir l&#39;ouverture de la fiole avec un morceau de papier d&#39;aluminium. </li><li> Stériliser la solution à l&#39;autoclave à des conditions standards (121 psi, 120 ° C, 20 min). </li><li> Laisser la solution refroidir à 45 ° C dans un bain d&#39;eau. </li><li> Pour la solution, ajouter ce qui suit dans l&#39;ordre: 500 cholestérol ul de 5 mg / ml de solution mère (dissous dans l&#39;éthanol), 500 pi 1 M CaCl 2, 500 pi 1 M MgSO 4, et 12,5 ml 4 tampon KPO (54,15 g KH 2 PO 4 et de 17,8 g de K 2 HPO 4 dans 500 ml de dH 2 O) comme décrit dans 18. </li><li> L&#39;aide de pipettes stériles, ajouter 10 ml de solution d&#39;agar à chaque 60 mm de polystyrène boîte de Pétri. Autoriser les médias à se solidifier à la température ambiante overnight. </li></ol> 2) Préparation du bouillon B E. Médias coli <ol><li> Pour faire cinq cultures de 100 ml, ajouter 5,0 g de Bactotryptone, 2,5 g d&#39;extrait de levure, 5,0 g NaCl dans un bécher. </li><li> Ajouter de l&#39;eau distillée stérile pour un volume final de 500 ml. Chauffer la solution sur une plaque chaude, sous agitation, jusqu&#39;à ce que les solutés sont dissous. </li><li> Versez 100 ml de bouillon B en cinq bouteilles de verre (ml volume au moins 200). </li><li> Stériliser les solutions par autoclavage à des conditions standard (121 psi, 120 ° C, 20 minutes). </li><li> Laissez B bouillon refroidir à température ambiante avant utilisation. </li></ol> 3) L&#39;ensemencement C. elegans Plaques d&#39;entretien <ol><li> Après avoir laissé les plaques NGM sécher à température ambiante pendant au moins une semaine, inoculer un B 100 ml bouillon de culture avec quelques colonies de E. coli (OP50). </li><li> Croître E. culture coli à 37 ° C pendant une nuit. </li><li> Pipette une à deux gouttes (environ 10081; l) de OP50 culture sur le centre de chaque plaque NGM. Les plaques sont généralement empilées couvercle vers le haut. Lorsque pipetage (ensemencement), commencer par la pipette de la culture sur la plaque au fond de la pile. Hissez-vous chaque pile. Essayez de ne pas déplacer les plaques après le semis, car il est important que la culture soit pas trop près des bords de la plaque. </li><li> Laisser les plaques ensemencées sécher à température ambiante pendant au moins une semaine avant de l&#39;utiliser. </li><li> Plaques ensemencées sont stockés (inversé) dans des récipients en plastique scellés à température ambiante. </li></ol> 4) Maintien C. elegans Souches <ol><li> Pour maintenir bien nourris C. les stocks elegans, un ver utilisent choisir de passer trois L4s ou adultes gravides à une nouvelle tête de série NGM plaque tous les 3-4 jours. </li><li> plaques de magasins (inversée) de préférence à l&#39;intérieur d&#39;un incubateur à 15-22 ° C. Cultures dans les analyses décrites ont été conservés à 20 ° C. C. développement elegans est dépendante de la température. Comme maintenance augmentation de la température, le temps entre les stades de développement diminue. </li></ol> 5) Préparation de soja tryptique Agar (TSA) E. faecalis Culture Médias <ol><li> Pour faire 50 plaques, mesurer 500 ml d&#39;eau distillée stérile dans un ballon et porter à ébullition en remuant, sur une plaque chauffante. </li><li> Ajouter 20 g de poudre de TSA agar dans le ballon et laisser la solution pour dissoudre. </li><li> Stériliser les solutions par autoclavage à des conditions standard (121 psi, 120 ° C, 20 minutes). </li><li> Laisser la solution refroidir à 45 ° C dans un bain d&#39;eau. </li><li> L&#39;aide de pipettes stériles, ajouter 10 ml de solution d&#39;agar à chaque 60 mm de polystyrène boîte de Pétri. Autoriser les médias à se solidifier à température ambiante jusqu&#39;au lendemain. </li></ol> 6) Préparation de soja tryptique Broth (TSB) E. faecalis Culture Médias <ol><li> Pour faire 250 tubes de culture, mesurer 500 ml d&#39;eau distillée stérile dans un flacon et chaud sur une plaque de cuisson en remuant. </li&gt; <li> Ajouter 15 g de poudre d&#39;agar TSB dans le ballon et laisser la poudre à dissoudre. </li><li> Stériliser les solutions par autoclavage à des conditions standard (121 psi, 120 ° C, 20 minutes). </li><li> Laisser refroidir la solution à température ambiante, puis pipette 2 ml, 5 dans des tubes à essai stériles ml. </li></ol> 7) Préparation de la perfusion (BHI) Médias coeur du cerveau, avec la streptomycine, pour E. faecalis Culture <ol><li> Mesurer 500 ml d&#39;eau distillée stérile dans un ballon et porter à ébullition en remuant, sur une plaque chauffante. </li><li> Ajouter 26 g de milieu BHI dans le ballon et laisser la solution pour dissoudre. </li><li> Stériliser les solutions par autoclavage à des conditions standard (121 psi, 120 ° C, 20 minutes). </li><li> Laisser refroidir la solution à 45 ° C dans un bain d&#39;eau. </li><li> En utilisant une technique stérile, ajouter 0,5 ml d&#39;une solution mère de streptomycine 10 mg / ml et agiter pour mélanger. </li><li> Ajouter 10 ml de la solution à chaque 60 mm de polystyrène boîte de Pétri. </li><li&gt; Autoriser les médias à se solidifier à température ambiante jusqu&#39;au lendemain. plaques de magasins (inversée) à 4 ° C jusqu&#39;à ce que quelques heures avant l&#39;utilisation. </li></ol> 8) Le maintien E. faecalis souches <ol><li> Pour maintenir E. les stocks faecalis, utilisez une boucle stérile traînée colonies individuelles sur gélose plaques de soja tryptique distincts (TSA). </li><li> Placer les cultures à 37 ° C pendant la nuit. plaques de magasins (inversé) dans un sac scellé ou boîte à température ambiante pendant plusieurs semaines. </li></ol> 9) Préparation E. Plaques faecalis pour EIW Assay <ol><li> Inoculer une culture 2 ml TSB avec une colonie de E. faecalis et incuber une nuit à 37 ° C. </li><li> Pipette 20 ul de E. culture faecalis sur le centre d&#39;une plaque BHI-streptomycine préparé, et incuber une nuit à 37 ° C. </li></ol> 10) Préparation E. Plaques coli pour EIW Assay (plaques de contrôle) <ol><li> Inoculer un ml B-bro 100e culture avec une colonie de E. coli OP50 et incuber une nuit à 37 ° C </li><li> Pipette 20 ul de la culture OP50 sur le centre d&#39;une plaque non ensemencée NGM et permettent aux plaques ensemencées sécher à température ambiante jusqu&#39;au lendemain. </li></ol> 11) Egg essai de Worm <ol><li> Choisissez de 15 à 20 en scène-L4 C. elegans (Figure 1) au centre d&#39;un tapis de bactéries sur un BHI-Strep-E. préparé plaque faecalis. Veillez à ne pas transférer beaucoup de OP50 à la plaque BHI. Choisissez le même nombre de L4s à un contrôle NGM-OP50 plaque. </li><li> Incuber les plaques pendant 40 heures à 20 ° C. </li><li> Préparer une solution d&#39;eau de Javel à 20%. Mélangez une eau de Javel commerciale (6,0% d&#39;hypochlorite de sodium) avec le volume approprié d&#39;eau distillée. </li><li> Ajoutez 10 gouttes ul d&#39;une solution de javel pour dix emplacements distincts sur un couvercle en plastique (d&#39;un ver ou d&#39;une plaque bactérienne). </li><li> Avec une pointe de ver, transférer un ver dans chaque goutte d&#39;eau de Javel. Remuer doucement la pointe de l&#39;choisir dans la gouttelette eau de Javel pour s&#39;assurer que le ver a lavé la fin de la pioche (confirmer en regardant la goutte sous une loupe binoculaire). </li><li> Laisser la cuticule de dissoudre environ 10 min ou jusqu&#39;à ce que les vers éclatent, en expulsant les oeufs (figure 2). Veillez à ne pas transférer les oeufs de la plaque. </li><li> En utilisant un microscope à dissection, de compter les œufs dans chaque goutte d&#39;eau de Javel. </li></ol>

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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Les étapes les plus importantes dans l&#39;accomplissement avec succès ce test sont les suivants: 1) l&#39;utilisation des stocks bien nourris de C. elegans, 2) la culture des types individuels de bactéries sur la plaque de dosage, 3) l&#39;identification précise scène L4 vers l&#39;exposition à E. faecalis, 4) en maintenant la durée d&#39;exposition à E. faecalis uniformes dans tous les essais et 5) le temps de blanchiment ne doivent pas dépasser dix minutes pour éviter la désinté...

Caenorhabditis elegans, un microscopique, ascaris vivant en liberté, est un organisme modèle traditionnellement utilisé pour étudier les processus de signalisation du développement et de la cellule en raison de son anatomie transparent, développement bien caractérisé, entièrement séquencé génome, la génération en temps court, et l&#39;homologie génétique à l&#39;homme . Plus récemment, C. elegans est devenu un organisme modèle dans le domaine de la toxicologie environnementale et l&#39;immunité innée 10, 11. Ces auto-fertilisation vers hermaphrodites deviennent sexuellement matures dans les deux à trois jours après l&#39;éclosion de l&#39;oeuf. Au cours de son cycle de vie, C. elegans passe par quatre stades larvaires (L1-L4), avant d&#39;atteindre l&#39;âge adulte. Un isolé hermaphrodite peut produire, en moyenne, 300 enfants dans les trois jours de fécondité maximale. En sexuellement matures C. elegans hermaphrodite, les œufs fécondés sont retenus dans l&#39;utérus pendant plusieurs heures avant d&#39;être jeté. Lenombre normal des oeufs stockées dans la matrice à un moment donné (en période de pointe fécondité) est comprise entre dix et quinze 12. Le nombre d&#39;oeufs dans l&#39;utérus est une fonction à la fois le taux de production d&#39;oeufs et le taux de ponte. Les œufs fécondés sont expulsés de l&#39;utérus par la contraction des muscles seize vulvaires disposées autour de l&#39;ouverture de la vulve 13. Motoneurones Hermaphrodite spécifiques (HSN départ) et les motoneurones VC synapse sur les muscles vulvaires affectant la contraction musculaire et donc la ponte comportement 5,7,13,14. Expulsion des œufs de l&#39;utérus est dû à l&#39;activité coordonnée de neurones et les muscles. cultures de laboratoire de C. elegans sont typiquement élevés à un régime de non pathogène Escherichia coli OP50. Dans le milieu naturel, C. elegans entrent en contact avec une variété de sources alimentaires, tels que les bactéries pathogènes, qui peuvent être potentiellement dangereux. Lorsqu&#39;ils sont exposés à des substances nocives enl&#39;environnement, C. elegans conserver les oeufs jusqu&#39;à ce que l&#39;environnement devient plus favorable. On peut supposer que cette rétention d&#39;œuf est un effort pour protéger leur progéniture. Dans cet œuf à vis sans fin (EIW) dosage, C. elegans sont exposés aux bactéries pathogènes potentiellement, Enterococcus faecalis, qui se trouve dans l&#39;environnement. L&#39;exposition à des formes pathogènes de E. faecalis peuvent causer une infection intestinale persistante et même la mort en C. elegans 15. L&#39;exposition à d&#39;autres formes de bactéries pathogènes ont été montré pour affecter la rétention d&#39;œuf 16,17, cependant l&#39;effet n&#39;a pas été quantifiée. En outre, les souches de l&#39;effet de légèrement pathogène de E. faecalis, les souches qui ne sont pas immédiatement mortelle, sur le comportement de ponte n&#39;a pas été étudiée. Dosages EIW impliquent compter le nombre d&#39;œufs conservés dans l&#39;utérus de C. elegans 4. Même si C. eleganssont transparentes, les œufs s&#39;accumulent dans l&#39;utérus peut être difficile à quantifier dans un animal intact. Le test consiste à EIW blanchiment gravide adulte C. elegans qui ont été exposées à la bactérie pendant une période de temps déterminée. La solution d&#39;eau de Javel dissout la cuticule externe laissant les œufs derrière. Les oeufs sont réfraction aux effets de blanchiment en raison de la présence d&#39;une coquille de protection. Après le blanchiment, on est très facilement en mesure de compter le nombre d&#39;œufs libérés de l&#39;utérus des vers sur le blanchiment. L&#39;essai décrit une méthode simple, peu coûteux et rapide à quantifier le nombre d&#39;ovules dans l&#39;utérus à un moment donné, et donc de quantifier les effets de E. faecalis sur la rétention d&#39;œufs. Ce test peut être utilisé pour quantifier l&#39;effet d&#39;autres types de bactéries, de toxines environnementales ou des médicaments sur la rétention d&#39;œufs. Ce test a également le potentiel d&#39;être utilisé comme un écran pour pouvoir pathogène bactérien.

<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. comportement de ponte elegans est affecté par des signaux environnementaux tels que l&#39;osmolarité 1 et vibrations 2. En l&#39;absence totale de nourriture C. elegans cessent également de ponte et de conserver les œufs fécondés dans l&#39;utérus 3. Cependant, l&#39;effet des différentes sources de nourriture, en particulier les bactéries pathogènes et notamment Enterococcus faecalis, sur le comportement de ponte n&#39;est pas bien caractérisée. L&#39;œuf en ver essai (EIW) est un outil utile pour quantifier les effets de différents types de bactéries, dans ce cas E. faecalis, sur le comportement de ponte. Dosages EIW impliquent compter le nombre d&#39;œufs conservés dans l&#39;utérus de C. elegans 4. Le test consiste à EIW blanchiment mis en scène, gravide adulte C. elegans pour enlever la cuticule et séparent les œufs conservés provenant de l&#39;animal. Avant de blanchiment, les vers sont exposés à des bactéries (ou tout autre type de signal environnemental) Pendant une période de temps déterminée. Après le blanchiment, on est très facilement en mesure de compter le nombre d&#39;œufs conservés à l&#39;intérieur de l&#39;utérus des vers. Dans cet essai, une augmentation quantifiable de la rétention d&#39;œuf après E. exposition faecalis peut être facilement mesuré. Le test EIW est un test comportemental qui peut être utilisé pour le dépistage de bactéries potentiellement pathogènes ou la présence de toxines dans l&#39;environnement. En outre, le test EIW peut être un outil pour dépister les drogues qui affectent neurotransmetteur signalisation depuis comportement de ponte est modulée par des neurotransmetteurs comme la sérotonine et l&#39;acétylcholine 5-9.

Ce test permet de quantifier le nombre d&#39;œufs retenus dans C. elegans après exposition à E. faecalis. L4 scène vers (caractérisé par l&#39;espace ouvert et transparent au-dessus de la vulve, figure 1) ont été exposés à E. faecalis l&#39;âge adulte. Après 40 heures d&#39;exposition à E. faecalis, le blanchiment a été réalisée. Comme la cuticule désintégré dans la goutte d&#39;eau de javel, les œufs sont devenus plus apparents (figure 2)...

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 test d&#39;oeuf en ver (EIW) est une méthode utile pour quantifier comportement de ponte. Altérations de la ponte peut être une réponse comportementale de l&#39;organisme modèle<em&gt; Caenorhabditis elegans</em&gt; À des substances environnementales potentiellement nocives telles que celles produites par des bactéries pathogènes.

Mesurer les effets des bactéries sur<em&gt; C. Elegans</em&gt; Comportement aide d&#39;un test de rétention d&#39;oeufs

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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16.9K vues.  Fairleigh Dickinson University. Un test d'oeuf en ver (EIW) est une méthode utile pour quantifier comportement de ponte. Altérations de la ponte peut être une réponse comportementale de l'organisme modèle Caenorhabditis elegans À des substances environnementales potentiellement nocives telles que celles produites par des bactéries pathogènes.

Vidéo: 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.

Génération de Stable elegans transgéniques C. Utilisation de microinjection

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

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Biologie

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.

Mesure Caenorhabditis elegans</emDurée de vie> sur des milieux solides

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.

Imagerie C. elegans Les embryons en utilisant un microscope à épifluorescence et de logiciels Open Source

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.

Visualisation de la fonction respiratoire mitochondriale utilisant Cytochrome C Oxydase / succinate déshydrogénase (COX / SDH) Double-histochimie étiquetage

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

Création d&#39;environnements gazeux définies pour étudier les effets de l&#39;hypoxie sur la<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 test pour évaluer le comportement des explants cellulaires du mésenchyme crânienne

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.

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.

Anticorps coloration dans<em&gt; C. Elegans</em&gt; Utilisation de «gel-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.

À grande échelle Gene Knockdown dans<em&gt; C. elegans</em&gt; Utilisation ARNdb alimentation bibliothèques pour générer des phénotypes robustes perte de fonction

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 modèle anoxie-faim pour ischémie / reperfusion dans<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 ...