Yazarlar E. tedarik Haziran Middleton teşekkür etmek istiyorum faecalis suşları ve rehberlik için bakteri kültürü ile. C. elegans Araştırma Altyapısı Programları NIH Ofisi (P40 OD010440) tarafından finanse edilmektedir CGC, tarafından verilmiştir.

Nematod Büyüme Medya 1) hazırlanması (NGM) <ol><li> 50 levha yapmak için, 1.5 g NaCl, 8.5 g saf olan Ağar ve 1 Lflask 1.25 g pepton ekleyin. </li><li> Balona dH 2 O 487.5 ml ekleyin. Alüminyum folyo bir parça balonun açılış karıştırmak ve karşılamak için yavaşça girdap. </li><li> Standart koşullar (121 psi, 120 ° C, 20 dakika) otoklav ile çözüm sterilize edin. </li><li> Çözüm bir su banyosunda 45 ° C soğumasını bekleyin. </li><li> 5 mg / ml stok çözeltisi (etanol içinde çözülmüş), 500 ul 1 M CaCI2, 500 ul 1 M MgSO 4 ve 12.5 ml KPO 4 tampon maddesi (54.15 g ile 500 ul: kolesterol çözeltisine, sırayla aşağıdaki eklemek dH 2 O 500 ml KH 2 PO 4 ve 17.8 g K 2 HPO 4) 18 olarak tanımlanmıştır. </li><li> Steril pipetler kullanarak, her 60 mm polistiren Petri kabı agar çözüm 10 ml ekleyin. Medya oda sıcaklığında overn katılaşmaya izinight. </li></ol> B Broth E. 2) hazırlanması coli Medya <ol><li> Beş, 100 ml kültür hale getirmek için, bir behere Bacto Tripton, 2.5 g maya özütü, 5.0 g NaCl, 5.0 g ekleyin. </li><li> 500 ml &#39;lik bir son hacim için steril, damıtılmış su ekleyin. Çözünen çözünmüş kadar, karıştırarak, sıcak bir plaka üzerinde çözüm ısıtın. </li><li> Beş cam şişe (en az 200 ml hacim) içine Oda suyu 100 ml dökün. </li><li> Standart koşullar (121 psi, 120 ° C, 20 dakika) otoklav ile çözüm sterilize edin. </li><li> B suyu kullanmadan önce oda sıcaklığına kadar soğumasını bekleyin. </li></ol> 3) Tohumlama C. elegans Bakım Tabaklar <ol><li> NGM plakalar en az bir hafta için oda sıcaklığında kuruması için izin verdikten sonra, E. birkaç koloni ile bir 100 ml oda suyu kültürü aşılamak coli (OP50). </li><li> E. büyütün 37 ° C&#39;de gece boyunca kültür coli. </li><li> Pipet bir-iki damlası (yaklaşık 10081, l) OP50 kültürünün her NGM plaka ortasına üzerine. Tabaklar genellikle kapak tarafı yukarı dizilir. (Ekim) pipetleme zaman, yığının altındaki plaka üzerine kültür pipetleme başlar. Her yığın şekilde çalışır. Bu kültür çok yakın plakanın kenarlarına alamadım önemlidir çünkü, tohumlama sonra plakaları taşımak için çalışın. </li><li> Tohumlanan plakalar kullanılarak önce en az bir hafta için oda sıcaklığında kurumaya bırakın. </li><li> Tohumlu plakalar oda sıcaklığında kapalı plastik kaplarda (ters) saklanır. </li></ol> 4) C. bakımı elegans Suşlarının <ol><li> Iyi beslenen C. korumak için elegans stokları, her 3-4 günde bir yeni bir, numaralı seribaşı NGM plaka için üç L4S veya gebe yetişkin taşımak için almak bir solucan kullanın. </li><li> Mağaza plakalar (ters) tercihen bir inkübatör içinde 15 - 22 ° C Açıklanan tahlillerinde kültürler 20 ° C C de muhafaza edildi elegans geliştirme sıcaklığa bağlıdır. Maintena gibisıcaklık artışları nce, gelişim evreleri arasında zaman azalır. </li></ol> Triptik soya ağarı (TSA) E. 5) hazırlanması faecalis Kültür Medya <ol><li> Bir ocak, karıştırarak 50 plaka yapmak için, bir balona steril, distile su 500 ml ölçmek ve bir taşım kaynatın. </li><li> Şişeye toz TSA agar 20 g ilave edin ve eritmek için bir çözüm sağlar. </li><li> Standart koşullar (121 psi, 120 ° C, 20 dakika) otoklav ile çözüm sterilize edin. </li><li> Çözüm bir su banyosunda 45 ° C soğumasını bekleyin. </li><li> Steril pipetler kullanarak, her 60 mm polistiren Petri kabı agar çözüm 10 ml ekleyin. Ortam gece boyunca oda sıcaklığında katılaşmaya izin verin. </li></ol> Tryptic Soy Broth (TSB) E. 6) hazırlanması faecalis Kültür Medya <ol><li> Karıştırılırken 250 kültür tüpleri hale getirmek için, bir ocak üzerindeki bir şişeye ve sıcak olarak, steril, damıtılmış su, 500 ml ölçer. </li&gt; <li> Şişeye TSB, agar tozu 15 g ekleyin ve tozu çözmek için izin verir. </li><li> Standart koşullar (121 psi, 120 ° C, 20 dakika) otoklav ile çözüm sterilize edin. </li><li> Steril, 5 ml test tüplerine çözelti oda sıcaklığına soğumaya bırakın ve daha sonra pipet 2 mi. </li></ol> 7) E. için Streptomisin ile Beyin Kalp İnfüzyon (BHI) Medya, hazırlanması, faecalis Kültür <ol><li> Bir şişe içine steril damıtılmış su, 500 ml ölçmek ve bir ocak, karıştırılırken, bir kaynama noktasına getir. </li><li> Şişeye BHI ortamı 26 g ekleyin ve çözelti, çözünmesi için izin verir. </li><li> Standart koşullar (121 psi, 120 ° C, 20 dakika) otoklav ile çözüm sterilize edin. </li><li> Çözüm ° C su banyosunda 45 soğumasını bekleyin. </li><li> Steril tekniği kullanarak, bir 10 mg / ml streptomisin stok solüsyonu ve karıştırmak için girdap 0.5 ml. </li><li> Her 60 mm polistiren Petri kabı çözüm 10 ml ekleyin. </li><li&gt; Veren ortam gece boyunca oda sıcaklığında katılaşmaya bırakılır. Mağaza, 4 tabak (ters) ° kullanmadan önce birkaç saat kadar C. </li></ol> 8) E. bakımı faecalis suşları <ol><li> E. korumak için faecalis hisse senetleri, ayrı triptik soya agar (TSA) plakalar üzerine çizgi tek tek koloniler steril bir döngü kullanın. </li><li> 37 ° C gecede kültür büyütün. Birkaç hafta boyunca oda sıcaklığında kapalı bir torba ya da kutu içinde plakları (ters). </li></ol> 9) E. hazırlanması EIW Testi için faecalis Plakalar <ol><li> E. bir koloni ile 2 ml TSB kültürü aşılamak faecalis ve 37 gece boyunca inkübe ° C </li><li> E. Pipet 20 ul faecalis hazırlanmış BHI-streptomisin plakasının ortasına üzerine kültür ve 37 gece boyunca inkübe ° C </li></ol> 10) E. hazırlanması EIW Testi (Kontrol Levhalar) için coli Tabaklar <ol><li> 100 ml&#39;lik bir B-bro inoküleE. bir koloni ile inci kültürü coli OP50 ve 37 ° C gece boyunca inkübe </li><li> Pipet 20 bir giremeyen NGM plaka merkezi üzerine OP50 kültür ul ve tohumlu plakalar gece boyunca oda sıcaklığında kurumaya bırakın. </li></ol> Solucan Deneyi 11) Yumurta <ol><li> 15 almak - 20 sahnelenen-L4 C. hazırlanmış bir BHI-Strep-E. bakteri çim ortasında üzerine elegans (Şekil 1) faecalis plaka. BHI plakasına OP50 bir sürü aktarmak için dikkatli olun. NGM-OP50 plaka bir kontrol için L4S aynı sayıda seçin. </li><li> 20 ° C&#39;de 40 saat boyunca inkübe edin </li><li> 20% çamaşır suyu çözeltisi hazırlayın. Damıtılmış su, uygun hacmi ile ticari bir çamaşır suyu (% 6.0 sodyum hipoklorit) karıştırın. </li><li> Bir plastik kapak on farklı yerlerde (bir solucan veya bakteriyel plaka) için çamaşır suyu 10 ul damla ekleyin. </li><li> Bir solucan seçim kullanarak, her çamaşır suyu damla içine bir solucan aktarın. Bir Yavaşça girdap ucusolucan seçim sonunda (bir mikroskop altında damlacık görüntüleyerek teyit) yıkanır emin olmak için çamaşır suyu damlacık içinde almak. </li><li> Solucanlar yumurta (Şekil 2) kovma, açık patlayana kadar manikür yaklaşık 10 dakika ya da çözmek için izin verin. Plaka yumurta transferi için dikkatli olun. </li><li> Bir mikroskop kullanarak, çamaşır suyu her damla yumurta sayısı. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarının olmadığını beyan ederim.

Başarılı bir şekilde bu testte performans en kritik adımlar şunlardır: 1) C iyi beslenmiş desteleri kullanarak, tahlil plaka, 3 elegans, 2) kültür bakteri tek tip) doğru belirlenmesi E. maruz kalma L4 solucanlar sahnelenen faecalis, 4) E. maruz kalma süresi tutmak zaman ağartma tüm çalışmalar ve 5 tutarlı faecalis) yumurta parçalanma önlemek için on dakika geçmemelidir. Bu testi için sağlıklı, iyi beslenmiş solu...

Caenorhabditis elegans, mikroskobik, serbest yaşayan yuvarlak kurt, geleneksel olarak insanlara nedeniyle şeffaf anatomi, iyi karakterize geliştirme, tam sıralı genom, kısa nesil zamanlı ve genetik homoloji gelişim ve hücre sinyal süreçleri incelemek için kullanılan bir model organizmadır . Daha yakın zamanda, C. elegans çevresel toksikoloji ve doğuştan gelen bağışıklık 10, 11 alanında bir model organizma haline gelmiştir. Bu kendi kendine gübreleme hermaphroditic solucanlar yumurtadan çıkım iki-üç gün içinde cinsel olgun olur. Yaşam döngüsü, C. sırasında elegans yetişkinliğe ulaşmadan önce, dört larva aşamaları (L1-L4) geçer. Bir izole hermafrodit tepe verimlilik üç gün içinde, ortalama, 300 yavru üretebilir. Üreme olgun C. elegans çift cinsiyetli, döllenmiş yumurta koydu önce birkaç saat boyunca rahim içinde korunur.Herhangi bir anda, rahim içinde depolanan yumurta normal sayıda (en yüksek verimlilik esnasında) on ila onbeş 12&#39;dir. Rahim yumurta sayısı yumurta üretimi oranı ve yumurtlama oranını hem de bir fonksiyonudur. Döllenmiş yumurta vulva 13 açılış etrafında düzenlenmiş on altı vulva kasların kasılması ile rahim kesilir. Hermafrodit özel nörotoksinlerini (HSN) &#39;ın ve dolayısıyla VC nörotoksinlerini kas kasılması etkileyen vulva kasları üzerine sinaps ve yumurtlama davranışı 5,7,13,14. Rahim yumurta ihraç nöronlar ve kasların koordine aktivite nedeniyle oluşur. C. Laboratuar kültürleri elegans genellikle patojen olmayan Escherichia coli OP50 bir diyet yetiştirilir. Doğal ortamda, C. elegans gibi patojen bakteriler gibi gıda kaynakları, çeşitli temas, potansiyel olarak zararlı olabilir. Zararlı maddelere maruz kaldığındaçevre, C. çevreye daha elverişli hale gelinceye kadar elegans yumurta saklayın. Muhtemelen bu yumurta tutma onların soyu korumak için bir çabadır. Solucan (EIW) testi, C bu yumurta elegans, potansiyel olarak patojenik bakteriler, ortamda bulunan Enterococcus faecalis, maruz kalmaktadır. E. patojenik biçimlerine maruz faecalis C. kalıcı bağırsak enfeksiyonu ve hatta ölüme neden olabilir elegans 15. Patojenik bakterilerin diğer formlarına maruz kalma yumurta tutma 16,17 etkilediği gösterilmiştir, bununla birlikte etki miktarı değildi. E. Buna ek olarak, hafif patojenik etkisini suşları faecalis, yumurta döşeme davranış, hemen öldürücü olmayan suşları çalışılmamıştır. EIW deneyleri C rahim korudu yumurta sayısı, sayım içerir elegans 4. Olsa ° C. eleganssaydam, rahim içinde biriken yumurta bozulmamış bir hayvanda ölçmek için zor olabilir. EIW testi gebe yetişkin C. beyazlatma içerir zaman sabit bir süre için bakterilere maruz bırakıldı elegans. Çamaşır suyu solüsyonu geride bırakarak yumurta dış kütikül çözünür. Yumurta kabuğu koruyucu bir varlığı nedeniyle ağartıcı etkilerine refraktif vardır. Ağartma sonra, bir çok kolay beyazlatma üzerine solucanlar rahim salınan yumurta sayısını yapabiliyor. Bu deney, bir anda rahim içinde yumurtaların sayısını ölçmek için basit, ucuz ve hızlı bir yöntemdir, ve böylece E. etkilerini ölçmek yumurta tutma üzerinde faecalis. Bu testte yumurta tutma bakteri, çevresel toksinler veya ilaç diğer türleri etkisini ölçmek için kullanılabilir. Bu deney aynı zamanda bakteriyel patojen için bir ekran olarak kullanılmak üzere bir potansiyele sahiptir.

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		<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 yumurta döşeme davranış, osmolaritesi 1 ve titreşim 2 gibi çevresel ipuçları etkilenir. Gıda C. toplam yokluğunda elegans da yumurta döşeme ateşkes ve rahim 3 döllenmiş yumurta korumak. Ancak, özellikle farklı gıda kaynakları, özellikle patojen bakteri ve yumurta döşeme davranış Enterococcus faecalis, etkisi iyi karakterize değildir. Yumurta-in-solucan (EIW) testi bu durumda E&#39;de, bakteri farklı etkilerini ölçmek için bir araçtır faecalis, yumurta döşeme davranış. EIW deneyleri C rahim korudu yumurta sayısı, sayım içerir elegans 4. EIW testi sahnelenen ağartma, gebe yetişkin C. içerir elegans manikür kaldırmak ve hayvan muhafaza yumurta ayırmak için. Önce beyazlatma için, solucan bakteri (veya çevresel işaret her türlü maruz kalan) Zaman belirli bir süre için. Ağartma sonra, bir çok kolay solucanlar rahim içinde muhafaza yumurta sayısını yapabiliyor. E. sonra yumurta tutma Bu deneyde, ölçülebilir bir artış faecalis maruz kolaylıkla ölçülebilir. EIW tahlil patojenik bakteri veya çevresel toksinler varlığı açısından taranması için kullanılabilecek bir davranış deneyidir. Buna ek olarak, EIW testi yumurta döşeme davranış, serotonin ve asetilkolin 5-9 gibi nörotransmitterlerin tarafından modüle olduğu için sinyalizasyon nörotransmitter etkileyen ilaçlar için ekrana bir araç olabilir.

Bu testte bir C içinde muhafaza yumurta sayısı ölçmek için sağlar E. maruz kaldıktan sonra elegans faecalis. L4 solucanlar (kendi vulva, Şekil 1 üzerinde şeffaf açık alan ile karakterize) E. maruz bırakıldı sahnelenen yetişkinlik boyunca faecalis. E. maruz kalma kırk saat sonra faecalis, ağartma uygulandı. Kütikül ağartma damlacık içinde çözüldüler olarak, yumurta (Şekil 2) daha da belirgin hale geldi. M...

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

Bir yumurta-in-solucan (EIW) testi yumurtlama davranışı ölçmek için yararlı bir yöntemdir. Yumurtlama değişiklikler model organizma bir davranış tepki olabilir<em&gt; Caenorhabditis elegans</em&gt; Bu patojen bakteriler tarafından üretilen gibi zararlı çevresel maddelere.

Üzerinde Bakterilerin Etkilerinin Ölçme<em&gt; C. Elegans</emBir Yumurta Tutma Testi Kullanma&gt; Davranış

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 Görüntüleme.  Fairleigh Dickinson University. Bir yumurta-in-solucan (EIW) testi yumurtlama davranışı ölçmek için yararlı bir yöntemdir. Yumurtlama değişiklikler model organizma bir davranış tepki olabilir Caenorhabditis elegans Bu patojen bakteriler tarafından üretilen gibi zararlı çevresel maddelere.

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.

Mikroenjeksiyon Kararlı Transgenik C. elegans Üretimi

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

Biyoloji

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.

Ölçme Caenorhabditis elegans Katı Medya Ömrü

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.

Görüntüleme C. elegans Embriyolar Epifluorescent Mikroskop ve Açık Kaynak Yazılım

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.

Sitokrom kullanarak Mitokondriyal Solunum Fonksiyon Görselleştirme C Oksidaz / Succinat Dehidrogenaz (COX / SDH) Çift-etiketleme Histokimya

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

Üzerine Hipoksi Etkileri Eğitim için tanımlı Gazlı ortamlar oluşturma<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.

Kranial mesenchyme Hücresel Davranış Değerlendirme için bir Eksplantasyon Assay

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 Kemotaksis Deneyi

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.

Antikor boyamasında<em&gt; C. Elegans</em&gt; &quot;Freeze-Cracking kullanma&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.

Büyük ölçekli Gen demonte olarak<em&gt; C. elegans</em&gt; Sağlam Kayıp fonksiyon-fenotipleri oluşturmak için DsRNA Besleme Kitaplıklar kullanma

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.

Iskemi / reperfüzyon içinde bir anoksisi-açlık Modeli<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 ...