栽培<em&gt;秀丽隐杆线虫</em&gt;在三个维度实验室

摘要

我们提出了一个简单的方法来构建三维线虫培养系统称为NGT-3D和NGB-3D。这些可以被用来研究在更类似于天然线虫栖息比标准2D实验室线虫培养板栖息线虫健身和行为。

摘要

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.

引言

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 level^3-5.

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 matter^7,8. 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.

研究方案

1.准备NGT-3D和NGB-3D解决方案

1. 准备以下的无菌溶液:1升的0.1454摩尔NaCl溶液中，1升的1M的CaCl _2，1升的1M _硫酸镁的，LB培养基（LB），1升1M的KPO ₄缓冲液（108.3克KH ₂的PO ₄和35.6克K ₂的HPO _4，并填写H ₂ O操作1升）。利用这些解决方案NGM的生产可以在先前的协议¹⁰被发现。
2. 在121℃，15分钟的高压釜的解决方案。
3. 准备为50ml灭菌的5毫克/毫升胆固醇溶液。在50毫升的锥形管，混合0.25克胆固醇和50毫升99.99％乙醇拌匀。 不要高压 。使用50毫升注射器用0.45μm针筒式滤器灭菌胆固醇溶液中。这也将消除任何不溶解胆固醇。
4. （可选）准备的10ml 2'-脱氧-5-氟尿苷（的FUdR）股票150毫米的。在一个15毫升的锥形管，混合0.3693克的的FUdR和10ml蒸馏水。摇匀。

2.准备细菌培养为NGT-3D和NGB-3D

1. 接种10毫升LB培养基细菌。用于线虫喂食标准细菌大肠杆菌菌株OP50。
2. 培养在37℃下在振荡培养箱中过夜的细菌接种。
3. 在用于连续稀释制备，等分9毫升的NaCl溶液到无菌的15毫升锥形管中。例如，分装9毫升共打入7管做一个^10-7稀释OP50应变大肠杆菌 。
4. 使用无菌1000微升吸管，吸管将1ml细菌培养或稀释的细菌培养成育新管9毫升氯化钠溶液，涡旋10毫升混合好。重复，直到达到期望的细菌稀释。
   注意:所希望的细菌稀释取决于细菌的种类和条件以及确切的实验条件。例如，一个10 ^-6 - 10 ^-8稀释OP50菌株大肠杆菌的足以从1到生产-每6.5毫升NGT-3D管200的细菌菌落。蠕虫可靠发达国家和正常再生时的菌落数超过60，其在10 ^-6的稀释发生- ^10-7。对于NGB-3D，使用10 ^-8稀释。

3.使NGT-3D和NGB-3D（200ml）中

1. 制备细菌培养后，混合0.6克氯化钠，1g的造粒琼脂，和在一个500ml烧瓶0.5克胨。插入一个磁性搅拌棒的烧瓶中。
2. 添加195毫升蒸馏水和用铝箔覆盖该烧瓶的口。
3. 高压釜121℃15分钟。
4. 热高压灭菌烧瓶放置到一个搅拌盘，并以中等速度搅拌至少2小时。冷却烧瓶至40℃。一定要充分冷却从这里在高温下持续可能导致成品琼脂混浊。但是，较低的温度可能会导致在琼脂过早硬化。
   1. （可选）要加快冷却过程，将烧瓶在40℃水浴15分钟，放置在一个搅拌盘前。
5. 当琼脂培养基的温度达到40℃时，加入200微升的1M的CaCl _2，200微升5毫克/毫升胆固醇溶液，200微升的1M MgSO ₄上和5ml 1M的KPO ₄缓冲液作为溶液继续搅拌至1mM的CaCl _2，5微克/毫升胆固醇，1mM的MgSO ₄上，1mM的KPO ₄的最终浓度。
   1. （可选）NGT-3D寿命测定添加80μl的150mM的的FUdR的到120微米¹²的最终浓度。
6. 加6毫升10毫升稀释后直接从步骤2.4细菌培养成瓶的。
   1. （可选）对于NGT-3D，删除6毫升琼脂培养基步骤3.6之前保持温暖分开。此介质将用于细菌 - 自由顶层。
7. 使用无菌的程序，分配所述媒体到无菌的培养室。确保盒盖紧紧关闭。
   1. （可选的）为了防止细菌菌落从琼脂的顶部表面上形成，从步骤媒体3.7完全硬化之前在顶部从步骤3.6.1使无菌琼脂培养基的层。这将在NGT-3D的顶部创建一个无菌区。
   2. 为NGT-3D，倾6.5毫升介质插入8ml上述透明塑料试管，使细菌琼脂层，并小心地分配在半硬化3D媒体的顶部200微升无菌介质，以使薄无细菌在上面自由层。
   3. 对于NGB-3D，倒65毫升媒体到25 ^平方厘米的透明塑料细胞培养瓶。这一数额应填写25 ^平方厘米的细胞培养瓶的瓶体。
8. 离开垂直于室温室一周以允许细菌菌落生长到至少1毫米直径的具有相当规模。
   1. （可选）对于NGT-3D寿命试验，用铝箔盖室，以防止的FUdR的光降解。

在NGT-3D（相对育雏面积测定）蠕虫人口4.测量健身

1. 挑选使用铂丝的L4阶段蠕虫挑一个无细菌的NGM板转移。允许蠕虫自由走动几分钟，以去除附在它身上的细菌。
2. 重复步骤4.1和确保该蠕虫病毒是从细菌免费的。一般来说，4.1两个重复就足够了。
3. 小心地将清洁蜗杆与铂丝挑3D媒体的表面上。该蠕虫应最终进入琼脂进入3D琼脂矩阵。
4. 关上盖子松松地让一些空气进入管道，但防止琼脂培养基的干燥。
5. 在20℃孵育96小时。
6. 4天之后，紧紧地关闭盖子和放置NGT-3D培养室分成一个88℃的水浴以熔化的琼脂。热杀死虫子，但他们的身体保持不变。
   注意:使用8个毫升管的NGT-3D，20 - 30分钟潜伏期通常是足够的。
7. 用玻璃吸管，转移熔化的媒体到9厘米塑料培养皿。塑料吸管不建议，因为蠕虫病毒往往可以坚持给他们。
8. 使用透射立体声解剖显微镜，计数在L3，L4蠕虫和成年阶段的数目。不要指望那些L2级和年轻的蠕虫，作为F1和F2代可以在这里混淆。因此，这个实验是一个相对的育雏规模试验，而不是总育雏规模试验。

5.图像和NGB-3D记录蠕虫行为

1. 使用铂丝挑L4期蠕虫挑选并通过转移到一个无菌NGM板并使其自由几分钟移动除去粘在表面上的任何细菌。
2. 重复步骤5.1，以确保禾RM是细菌免费的。
3. 小心地将清洁蠕虫到NGB-3D的瓶用铂丝拾取的颈部附近的琼脂表面的中心。该蠕虫应最终进入琼脂进入3D琼脂矩阵。
4. 关上盖子松松地让一些空气进入管道，同时防止琼脂培养基的干燥。
5. 图片或录制立体声传输解剖显微镜下的蠕虫病毒。调整焦距为蠕虫移动通过3D矩阵。

结果

NGT-3D的结构是一个简单的和直接的协议，其导致在整个琼脂（ 图1A）间隔开小的细菌菌落在琼脂填充试管。蠕虫可通过琼脂矩阵自由移动，寻找和消费的细菌菌落。为了证实线虫是否可以复制和NGT-3D正常生长，我们比较土壤肥力与3D幼虫发育与标准的2D NGM板。在育雏相对大小分析，成年线虫在NGT-3D雌雄同体只是重现，以及在标准的2D NGM板雌雄同?...

讨论

采用经典的线虫的生长介质板线虫的实验室培养是至关重要的该研究线虫提供了数百个重要的发现。在这里，我们提出新的方法来培养线虫中更准确地反映其三维天然栖息地的环境。尽管其它方法已被用于在3D ¹³观察线虫 ，这是第一个协议，它允许蠕虫培养在固体三维基质。这里展示的两种方法，NGT-3D和NGB-3D，让科学家问3D养身健体，发展壮大的问题，也是图像...

材料

Name	Company	Catalog Number	Comments
LB broth, Miller (Luria-Bertani)	Difco	224620
Sodium chloride	DAEJUNG	7548-4400	58.44 MW
Agar, Granulated	Difco	214530
Peptone	Bacto	211677
Calcium chloride, dihydrate	Bio Basic	CD0050	2*H2O; 147.02 MW
Cholesterol	Bio Basic	CD0122	386.67 MW
Ethyl alcohol	B&J	RP090-1	99.99%; 46.07 MW
Magnesium sulfate, anhydrous	Bio Basic	MN1988	120.37 MW
Potassium phosphate, monobasic, anhydrous	Bio Basic	PB0445	136.09 MW
2'-Deoxy-5-fluorouridine	Tokyo Chemical Industry	D2235	246.19 MW
Potassium phosphate, dibasic, anhydrous	Bio Basic	PB0447	174.18 MW
Multi-Purpose Test Tubes	Stockwell Scientific	ST.8570	8 ml
Test Tube Closures	Stockwell Scientific	ST.8575
Cell Culture Flask	SPL Lifescience	70125	25 cm2
Research Stereo Microscope	Nikon	SMZ18
High-Definition Color Camera Head	Nikon	DS-Fi2
PC-Based Control Unit	Nikon	DS-U3
NIS-Elements Basic Research, Microscope Imaging Software	Nikon	MQS32000