Name: cultivation of caenorhabditis elegans in three dimensions in the laboratory
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עבודה זו נתמכה על ידי ניו החוקר גרנט [2014R1A1A1005553] מן קרן המחקר הלאומית של קוריאה (NRF) כדי ג&#39;יל; ומענק אתגר העתיד אוניברסיטת Yonsei המנהיג [2015-22-0133] כדי ג&#39;יל

1. הכינו פתרונות עבור NGT-3D ו NGB-3D <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את הפתרונות סטרילי הבאים: 1 ליטר של 0.1454 פתרון M NaCl, 1 ליטר של 1 M CaCl 2, 1 ליטר של 1 M MgSO 4, מרק Lysogeny (LB), 1 ליטר של 1 M KPO 4 חיץ (108.3 גרם של KH 2 4 PO ו 35.6 גרם של K 2 HPO 4, ולמלא H 2 O ל L 1). הפקה של NGM באמצעות פתרונות אלה ניתן למצוא בפרוטוקול קודם 10. </li><li style=";text-align:right;direction:rtl"> פתרונות חיטוי ב 121 ° C, 15 דקות. </li><li style=";text-align:right;direction:rtl"> הכן 50 מ&quot;ל של פתרון 5 מ&quot;ג / מ&quot;ל ​​כולסטרול סטרילי. בתוך צינור חרוטי 50 מ&quot;ל, לערבב 0.25 גרם של כולסטרול ו -50 מ&quot;ל של אתנול 99.99% ומערבבים היטב. אל חיטוי. לעקר את הפתרון כולסטרול באמצעות מזרק 50 מ&quot;ל עם מסנן מזרק 0.45 מיקרומטר. זה יהיה גם להסיר כל כולסטרול שלא נמס. </li><li style=";text-align:right;direction:rtl"> (אופציונלי) הכן 10 מ&quot;ל של 150 מ&quot;מ של 2&#39;-deoxy-5-fluorouridine המניות (FUDR). בתוך צינור חרוטי 15 מ&quot;ל, לערבב 0.3693 גרםשל FUDR ו -10 מ&quot;ל מים מזוקקים. נער היטב. </li></ol> 2. כן תרבות חיידקים עבור NGT-3D ו NGB-3D <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחסן 10 מ&quot;ל מרק LB עם חיידקים. החיידקים הסטנדרטיים המשמשים עבור אלגנס האכלה הוא OP50 זן החיידק. </li><li style=";text-align:right;direction:rtl"> תרבות חיסון החיידקים על 37 מעלות צלזיוס חממה רועדת הלילה. </li><li style=";text-align:right;direction:rtl"> לקראת דילול סדרתי, aliquot 9 מ&quot;ל של פתרון NaCl לתוך צינורות חרוטי 15 מ&quot;ל סטרילי. לדוגמה, aliquot 9 מ&quot;ל לתוך סך של 7 צינורות לבצע דילול 10 -7 של E. coli זן OP50. </li><li style=";text-align:right;direction:rtl"> בעזרת פיפטה 1,000 μl סטרילי, pipet 1 מ&quot;ל של תרבית החיידקים או התרבות חיידקים בדילול לתוך הצינור החדש סטרילי עם 9 מ&quot;ל של תמיסת NaCl ו מערבולת את התערובת 10 מ&quot;ל היטב. חזור על הפעולה עד דילול חיידקי הרצוי הוא הגיע. ההערה: דילול החיידקים הרצוי תלוי בסוג והמצב של חיידקים, כמו גם את תנאי ניסוי המדויקים.לדוגמה, 10 -6 - 10 -8 דילול של זן OP50 E. coli היה מספיק כדי להפיק מן 1 - 200 מושבות חיידקים לכל 6.5 מ&quot;ל צינור NGT-3D. תולעים שפותחו באופן מהימן ומתרבים בדרך כלל כאשר מספר מושבות היה מעל גיל 60, שאירע בשעת דילול של 10 -6 - 10 -7. לקבלת NGB-3D, השתמש דילול של 10 -8. </li></ol> 3. ביצוע NGT-3D ו NGB-3D (200 מ&quot;ל) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר הכנת תרבית החיידקים, לערבב 0.6 גרם NaCl, 1 גרם אגר מגורען, ו -0.5 גרם peptone בבקבוק 500 מ&quot;ל. הכנס בר ומערבבים מגנטיים לתוך הבקבוק. </li><li style=";text-align:right;direction:rtl"> להוסיף 195 מ&quot;ל מים מזוקקים לכסות את הפה של הבקבוק עם רדיד אלומיניום. </li><li style=";text-align:right;direction:rtl"> C חיטוי 121 מעלות במשך 15 דקות. </li><li style=";text-align:right;direction:rtl"> מניחים את הבקבוק autoclaved חם על צלחת ומערבבים, ומערבבים במהירות בינונית במשך שעה לפחות 2. מצננים את הבקבוק עד 40 ° C. הקפד לקרר מספיק כממשיך מכאן בטמפרטורות גבוהות עלול להוביל ערפול של אגר המוגמר.עם זאת, בטמפרטורות נמוכות עלולות לגרום התקשות המוקדמת של אגר. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> (אופציונלי) כדי להאיץ את תהליך הקירור, הנח את הבקבוק בתוך 15 דקות באמבט מים 40 ° C לפני הצבת בצלחת ומערבבים. </li></ol></li><li style=";text-align:right;direction:rtl"> כאשר הטמפרטורה של התקשורת אגר מגיע 40 ° C, להוסיף 200 μl 1 M CaCl 2, 200 μl של 5 מ&quot;ג / פתרון כולסטרול מ&quot;ל, 200 μl 1 M MgSO 4 ו -5 מ&quot;ל 1 M KPO 4 חיץ כפתרון ממשיכה לעורר לריכוזים סופי של 1 2 מ&quot;מ CaCl, 5 כולסטרול מיקרוגרם / מ&quot;ל, 1 מ&quot;מ 4 MgSO, 1 מ&quot;מ KPO 4. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> (אופציונלי) עבור assay תוחלת החיים NGT-3D להוסיף 80 μl של 150 מ&quot;מ FUDR לריכוז סופי של 120 מיקרומטר 12. </li></ol></li><li style=";text-align:right;direction:rtl"> הוסף 6 מ&quot;ל של 10 מ&quot;ל מדולל תרבית החיידקים משלב 2.4 לתוך הבקבוק ישירות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> (אופציונלי) עבור NGT-3D, להסיר 6 מ&quot;ל של התקשורת אגר לפני שלב 3.6 ו לשמור אותו חם בנפרד. מדיה זה ישמש את החיידקים ללאשכבה עליונה. </li></ol></li><li style=";text-align:right;direction:rtl"> בעזרת הליכים סטרילי, לוותר התקשורת לתוך תא תרבות סטרילי. ודא שהמכסה של החדר נסגרת בחוזקה. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> (אופציונאלי) כדי למנוע מושבות חיידקים מ טביעה על פני השטח העליונים של אגר, ליצור שכבה של תקשורת אגרה חיידקים ללא משלב 3.6.1 בחלק העליון בפני התקשורת משלב 3.7 מתקשה לחלוטין. פעולה זו תיצור אזור חיידקים ללא בחלק העליון של NGT-3D. </li><li style=";text-align:right;direction:rtl"> לקבלת NGT-3D, יוצקים 6.5 מ&quot;ל התקשורת לתוך מבחנות פלסטיק 8 מ&quot;ל ברור ליצור שכבה אגר חיידקים, ובזהירות לוותר 200 μl של התקשורת חיידקים חינם על גבי מדיה 3D קשוח למחצה לעשות bacteria- דק שכבה חינם על העליונה. </li><li style=";text-align:right;direction:rtl"> לקבלת NGB-3D, יוצקים 65 מ&quot;ל של המדיה לתוך בקבוק תרבית תאים 25 ס&quot;מ 2 פלסטיק שקוף. סכום זה צריך למלא את הגוף של בקבוק תרבות 25 ס&quot;מ 2 תאים. </li></ol></li><li style=";text-align:right;direction:rtl"> השאר תאים אנכיים בטמפרטורת חדר למשך שבוע כדי לאפשר מושבות חיידקים לגדוללגודל ניכר מ&quot;מ קוטר 1 לפחות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> (אופציונלי) עבור assay תוחלת החיים ב NGT-3D, תאי מכסים ברדיד אלומיניום כדי למנוע השפלה לאור FUDR. </li></ol></li></ol> 4. כושר מדוד אוכלוסין תולעת על NGT-3D (Assay גודל יחסית Brood) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פיק תולעת L4 לבמה בעזרת חוט פלטינה לקטוף העברה בצלחת NGM חיידקים חינם. אפשר תולעת לנוע בחופשיות במשך כמה דקות כדי להסיר חיידקים מחוברים לגוף שלה. </li><li style=";text-align:right;direction:rtl"> חזור על שלב 4.1 וודא כי התולעת היא ללא חיידקים. באופן כללי, ולשני שידורים חוזרים של 4.1 הם מספיק. </li><li style=";text-align:right;direction:rtl"> בזהירות במקום התולעת הנקיה על פני השטח של תקשורת 3D במכוש חוט פלטינה. התולעת צריכה בסופו של דבר להיכנס אגר לתוך המטריצה ​​אגר 3D. </li><li style=";text-align:right;direction:rtl"> סגור את המכסה רופף המאפשר אוויר כלשהו להגיע לתוך הצינור, אבל מניעת ההתייבשות של התקשורת אגרה. </li><li style=";text-align:right;direction:rtl"> דגירה של 96 שעות ב 20 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לאחר 4 ימים, לסגור את העפעפיים בחוזקה והמקוםNGT-3D תא תרבות לתוך אמבט מים 88 ° C כדי להמיס את אגר. החום הורג תולעים אך גופם נותרו על כנן. הערה: שימוש 8 מ&quot;ל צינורות עבור NGT-3D, 20 - 30 הדגירה דקות בדרך כלל מספיק. </li><li style=";text-align:right;direction:rtl"> בעזרת פיפטה זכוכית, להעביר את התקשורת מומסת על צלחת פטרי פלסטיק 9 ס&quot;מ. טפטפות פלסטיק לא מומלצות, כמו תולעים לעתים קרובות יכולים להידבק אליהם. </li><li style=";text-align:right;direction:rtl"> באמצעות מיקרוסקופ לנתח סטריאו שידור, לספור את מספר תולעים בבית L3, L4, ושלבי מבוגר. אל תסמכו תולעים כי הם בשלב L2 וצעיר, במרוצת הדורות F1 ו- F2 יכול להתבלבל כאן. לפיכך, assay זה הוא assay גודל להרהר ביחס ולא assay גודל גוזל הכולל. </li></ol> תמונה 5. והתנהגות תולעת שיא על NGB-3D <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> פיק תולעת L4 לבמה בעזרת חוט פלטינה לאסוף ולהסיר כל חיידקים תקועים אל פני השטח על ידי העברה לצלחת חיידקים ללא NGM ומאפשר לו לנוע בחופשיות במשך כמה דקות. </li><li style=";text-align:right;direction:rtl"> חזור על שלב 5.1 לוודא ווrm הוא ללא חיידקים. </li><li style=";text-align:right;direction:rtl"> בזהירות במקום התולעת הנקיה על גבי במרכז השטח אגר של NGB-3D ליד הצוואר של הבקבוק במכוש חוט פלטינה. התולעת צריכה בסופו של דבר להיכנס אגר לתוך המטריצה ​​אגר 3D. </li><li style=";text-align:right;direction:rtl"> סגור את המכסה רופף המאפשר אוויר כלשהו להגיע לתוך הצינור, תוך מניעת ההתייבשות של התקשורת אגרה. </li><li style=";text-align:right;direction:rtl"> תמונה או להקליט את התולעת תחת מיקרוסקופ לנתח סטריאו שידור. התאם את המיקוד כתולעת עוברת דרך מטריקס 3D. </li></ol>

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The authors have nothing to disclose.

טיפוח המעבדה של C. elegans באמצעות צלחות תקשורת צמיחת נמטודות הקלסיות היה חיוני למאות תגליות חשובות כי המחקר ב C. elegans ספקה. כאן, אנו מציגים שיטות חדשות לטפח סי אלגנס בסביבה בצורה מדויקת יותר משקפת גידול תלת מימדי הטבעי שלהם. למרות שיטות אחרות שימשו להתבונן <e...

The study of the nematode Caenorhabditis elegans in the laboratory has led to seminal discoveries in the field of biology over the last five decades1. C. elegans was the first multicellular organism to have its genome sequenced in 19982, and it has been invaluable in understanding the contributions of individual genes to the development, physiology, and behavior of a whole organism. Scientists now are looking to further understand how these genes may contribute to the survival and reproductive fitness of organisms in their natural environments, asking questions about ecology and evolution at the genetic level3-5. 
C. elegans once again can provide an excellent system to answer these questions. However, little is known about C. elegans biology in natural nematode habitats, and there are no current methods to simulate controlled natural conditions of C. elegans in the laboratory. In the lab, C. elegans is cultivated on the surface of agar plates seeded with E. coli bacteria6. In nature, however, C. elegans and related nematodes can be found sparsely inhabiting soils throughout the globe, but they are specifically found thriving in rotting fruits and vegetative matter7,8. These three-dimensional (3D) complex environments are quite different from the simple 2D environments to which worms are exposed to in the laboratory. 
To begin to answer questions about the biology of nematodes in a more natural 3D setting, we have designed a 3D habitat for laboratory cultivation of nematodes we called Nematode Growth Tube 3D or NGT-3D for short9. The goal was to design a 3D growth system that allows for comparable growth, development, and fertility to the standard 2D Nematode Growth Media (NGM) plates10. This system supports the growth of bacteria and nematodes over their entire life cycles in 3D, allows worms to move and behave freely in three dimensions, and is easy and inexpensive to manufacture and employ. 
In the current study, we provide a step-by-step method to manufacture NGT-3D and evaluate worm development and fertility. In addition to assessing worm fitness in 3D, we sought to image, video, and assess worm behavior and physiology in 3D cultivation. Thus, in addition to NGT-3D, we present here an alternate method called Nematode Growth Bottle 3D or NGB-3D, for the microscopic imaging of C. elegans during 3D cultivation. This will be especially important for the study of known behaviors identified in 2D, and the identification of novel behaviors unique to 3D cultivation.

<table><tbody><tr><td>LB broth, Miller (Luria-Bertani)</td><td>Difco</td><td>224620</td><td></td></tr><tr><td>Sodium chloride</td><td>DAEJUNG</td><td>7548-4400</td><td>58.44 MW</td></tr><tr><td>Agar, Granulated</td><td>Difco</td><td>214530</td><td></td></tr><tr><td>Peptone</td><td>Bacto</td><td>211677</td><td></td></tr><tr><td>Calcium chloride, dihydrate</td><td>Bio Basic</td><td>CD0050</td><td>2*H&lt;sub&gt;2&lt;/sub&gt;O; 147.02 MW</td></tr><tr><td>Cholesterol</td><td>Bio Basic</td><td>CD0122</td><td>386.67 MW</td></tr><tr><td>Ethyl alcohol</td><td>B&amp;amp;J</td><td>RP090-1</td><td>99.99%; 46.07 MW</td></tr><tr><td>Magnesium sulfate, anhydrous</td><td>Bio Basic</td><td>MN1988</td><td>120.37 MW</td></tr><tr><td>Potassium phosphate, monobasic, anhydrous</td><td>Bio Basic</td><td>PB0445</td><td>136.09 MW</td></tr><tr><td>2&#039;-Deoxy-5-fluorouridine</td><td>Tokyo Chemical Industry</td><td>D2235</td><td>246.19 MW</td></tr><tr><td>Potassium phosphate, dibasic, anhydrous</td><td>Bio Basic</td><td>PB0447</td><td>174.18 MW</td></tr><tr><td>Multi-Purpose Test Tubes</td><td>Stockwell Scientific</td><td>ST.8570</td><td>8 ml</td></tr><tr><td>Test Tube Closures</td><td>Stockwell Scientific</td><td>ST.8575</td><td></td></tr><tr><td>Cell Culture Flask</td><td>SPL Lifescience</td><td>70125</td><td>25 cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Research Stereo Microscope</td><td>Nikon</td><td>SMZ18</td><td></td></tr><tr><td>High-Definition Color Camera Head</td><td>Nikon</td><td>DS-Fi2</td><td></td></tr><tr><td>PC-Based Control Unit</td><td>Nikon</td><td>DS-U3</td><td></td></tr><tr><td>NIS-Elements Basic Research, Microscope Imaging Software</td><td>Nikon</td><td>MQS32000</td><td></td></tr></tbody></table>

cultivation of caenorhabditis elegans in three dimensions in the laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

בניית NGT-3D היא פרוטוקול פשוט וברור שתוצאתה הוא מבחנה המלאה אגרה עם מושבות חיידקים קטנות במרווחים לאורך (איור 1 א) אגר. תולעים יכולים לנוע בחופשיות דרך מטריקס אגר, מציאה וממצים את מושבות חיידקים. כדי לברר האם אלגנס יכולים להתרבות ולגדול ב...

Watch this Scientific Journal Video about Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory at JoVE.com

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

אנו מציגים שיטה פשוטה לבנות מערכות טיפוח נמטודות 3D נקרא NGT-3D ו NGB-3D. אלה יכולים לשמש כדי לחקור כושר נמטודות והתנהגויות בבתי גידול, כי הם יותר דומים לאזורים מחית elegans Caenorhabditis הטבעית מאשר צלחות תרבות elegans המעבדה ג 2D הסטנדרטיות.

טיפוח<em&gt; Elegans Caenorhabditis</em&gt; ב בשלושה ממדים במעבדה

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

cultivation-caenorhabditis-elegans-three-dimensions

Research

JoVE Journal

Biology

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

17.8K Views.  Yonsei University.  אנו מציגים שיטה פשוטה לבנות מערכות טיפוח נמטודות 3D נקרא NGT-3D ו NGB-3D. אלה יכולים לשמש כדי לחקור כושר נמטודות והתנהגויות בבתי גידול, כי הם יותר דומים לאזורים מחית elegans Caenorhabditis הטבעית מאשר צלחות תרבות elegans המעבדה ג 2D הסטנדרטיות. 

Video: Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

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

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

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

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

נמטודות. Caenorhabditis Elegans - מודל רב תכליתי In Vivo ללמוד אינטראקציות מארח-חיידק

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

Genetics

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple and inexpensive method to obtain a large number of animals for the isolation of proteins to proceed with a western blot, mass spectrometry, or further proteomics analyses. Furthermore, an increase of nematode numbers for immunostainings and the integration of multiple analyses under the same culturing conditions can easily be achieved. Additionally, a transfer between plates with different experimental conditions is facilitated. Common techniques in plate culture involve the transfer of a single C. elegans using a platinum wire and the transfer of populated agar chunks using a scalpel. However, with increasing nematode numbers, these techniques become overly time-consuming. This protocol describes the large-scale culture of C. elegans including numerous steps to minimize the impact of the sample preparation on the physiology of the worm. Fluid and shear stress can alter the lifespan of and metabolic processes in C. elegans, thus requiring a detailed description of the critical steps in order to retrieve reliable and reproducible results. C. elegans is a model organism, consisting of neuronal cells for up to one-third, but lacking blood vessels, thus providing the possibility to investigate solely neuronal alterations independent of vascular control. Recently, early neurodegeneration in diabetic retinopathy was found prior to vascular alterations. Thus, C. elegans is of special interest for studying general mechanisms of diabetic complications. For example, an increased formation of advanced glycation end products (AGEs) and reactive oxygen species (ROS) is observed, which are reproducibly found in C. elegans. Protocols to handle samples of adequate size for a broader spectrum of investigations are presented here, exemplified by the study of diabetes-induced biochemical alterations. In general, this protocol can be useful for studies requiring large C. elegans numbers and in which liquid culture is not suitable.

Plate-based Large-scale Cultivation of Caenorhabditis elegans: Sample Preparation for the Study of Metabolic Alterations in Diabetes

This protocol describes a method for the large-scale cultivation of Caenorhabditis elegans on solid media. As an alternative to liquid culture, this protocol allows obtaining parameters of different scales under plate-based cultivation. This increases the comparability of results by omitting the morphological and metabolic differences between liquid and solid media culture.

מבוסס על צלחת Large-scale טיפוח Caenorhabditis elegans: הכנת הדוגמא על המחקר של שינויים מטבוליים בחולי סוכרת

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Biochemistry

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans has emerged as one of the best models for studying development. From an optical point of view, its size and transparent body make this nematode an ideal specimen for DIC (Differential Interference Contrast or Nomarski) microscopy. This article illustrates a protocol for growing C. elegans nematodes, preparing and mounting their embryos, performing 4D microscopy and cell lineage tracing. The method is based on multifocal time-lapse records of Nomarski images and analysis with specific software. This technique reveals embryonic developmental dynamics at the cellular level. Any embryonic defect in mutants, such as problems in spindle orientation, cell migration, apoptosis or cell fate specification, can be efficiently detected and scored. Virtually every single cell of the embryo can be followed up to the moment the embryo begins to move. Tracing the complete cell lineage of a C. elegans embryo by 4D DIC microscopy is laborious, but the use of specific software greatly facilitates this task. In addition, this technique is easy to implement in the lab. 4D microscopy is a versatile tool and opens the possibility of performing an unparalleled analysis of embryonic development.

4D Microscopy: Unraveling Caenorhabditis elegans Embryonic Development Using Nomarski Microscopy

Here, we present a protocol for preparing and mounting Caenorhabditis elegans embryos, recording development under a 4D microscope and tracing cell lineage.

מיקרוסקופיה 4D: חשיפת קאנורהבדיטיס אלגנס התפתחות עוברית באמצעות מיקרוסקופיה של נומרסקי

4D microscopy is an invaluable tool for unraveling the embryonic developmental process in different animals. Over the last decades, Caenorhabditis elegans ...

Developmental Biology

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

בסיסי<em&gt; Caenorhabditis elegans</em&gt; שיטות: סנכרון תצפית

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

שבב מיקרופלואידי פשוט לגדילה והדמיה ארוכת טווח של Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding ...

Bioengineering

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

שיטה לCulturing עוברי<em&gt; ג elegans</em&gt; תאים

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Culturing<em&gt; Elegans Caenorhabditis</em &gt; בAxenic נוזלית מדיה ובריאה של תולעים מהונדסים ידי microparticle הפצצה

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these mechanisms is to introduce mutations in candidate genes and qualitatively describe changes in the morphology of tissues and cellular organelles. However, qualitative descriptions may not capture subtle phenotypic differences, might misrepresent phenotypic variations across individuals in a population, and are frequently assessed subjectively. Here, quantitative approaches are described to study the morphology of tissues and organelles in the nematode Caenorhabditis elegans using laser scanning confocal microscopy combined with commercially available bio-image processing software. A quantitative analysis of phenotypes affecting synapse integrity (size and integrated fluorescence levels), muscle development (muscle cell size and myosin filament length), and mitochondrial morphology (circularity and size) was performed to understand the effects of genetic mutations on these cellular structures. These quantitative approaches are not limited to the applications described here, as they could readily be used to quantitatively assess the morphology of other tissues and organelles in the nematode, as well as in other model organisms.

Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in Caenorhabditis elegans

This study outlines quantitative measurements of synaptic size and localization, muscle morphology, and mitochondrial shape in C. elegans using freely available image processing tools. This approach allows future studies in&#160;C. elegans&#160;to quantitatively compare the extent of tissue and organelle structural changes as a result of genetic mutations.

גישות כמותיים ללימוד מבנים סלולאריים ומורפולוגיה ארגונית בקנבאבדיוטיס

Defining the cellular mechanisms underlying disease is essential for the development of novel therapeutics. A strategy frequently used to unravel these ...

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid nematode growth media (NGM), allowing the efficient collection of population-level data for survival and other physiological phenotypes, and periodic sampling of subpopulations for fluorescent biomarker quantification. Limitations to this approach are the inability to (1) follow individual worms over time to develop age trajectories for phenotypes of interest and (2) monitor fluorescent biomarkers directly in the context of the culture environment. Alternative culture approaches use liquid culture or microfluidics to monitor individual animals over time, in some cases including fluorescence quantification, with the tradeoff that the culture environment is contextually distinct from solid NGM. The WorMotel is a previously described microfabricated multi-well device for culturing isolated worms on solid NGM. Each worm is maintained in a well containing solid NGM surrounded by a moat filled with copper sulfate, a contact repellent for C. elegans, allowing longitudinal monitoring of individual animals. We find copper sulfate insufficient to prevent worms from fleeing when subjected to aversive interventions common in aging research, including dietary restriction, pathogenic bacteria, and chemical agents that induce cellular stress. The multi-well devices are also molded from polydimethylsiloxane, which produces high background artifacts in fluorescence imaging. This protocol describes a new approach for culturing isolated roundworms on solid NGM using commercially available polystyrene microtrays, originally designed for human leukocyte antigen (HLA) typing, allowing the measurement of survival, physiological phenotypes, and fluorescence across the lifespan. A palmitic acid barrier prevents worms from fleeing, even in the presence of aversive conditions. Each plate can culture up to 96 animals and easily adapts to a variety of conditions, including dietary restriction, RNAi, and chemical additives, and is compatible with automated systems for collecting lifespan and activity data.

Long-Term Culture of Individual Caenorhabditis elegans on Solid Media for Longitudinal Fluorescence Monitoring and Aversive Interventions

Here, we present a protocol to culture isolated individual nematodes on solid media for lifelong physiological parameter tracking and fluorescence quantification. This culture system includes a palmitic acid barrier around single-worm wells to prevent animals from fleeing, allowing the use of aversive interventions, including pathogenic bacteria and chemical stressors.

תרבות ארוכת טווח של Caenorhabditis elegans בודדים על מדיה מוצקה לניטור פלואורסצנטי אורכי והתערבויות מרתיעות

Caenorhabditis elegans are widely used to study aging biology. The standard practice in C. elegans aging studies is to culture groups of worms on solid ...

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.

Long-Term Culture and Monitoring of Isolated Caenorhabditis elegans on Solid Media in Multi-Well Devices

Presented here is an optimized protocol for culturing isolated individual nematodes on solid media in microfabricated multi-well devices. This approach allows individual animals to be monitored throughout their lives for a variety of phenotypes related to aging and health, including activity, body size and shape, movement geometry, and survival.

תרבית ארוכת טווח וניטור של Caenorhabditis elegans מבודדים על מדיה מוצקה במכשירים מרובי בארות

The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, ...

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