定植 Euprymna scolopes鱿鱼由弧菌鲵</em
Summary
方法概括的过程，其中夏威夷短尾鱿鱼，<em> Euprymna scolopes</em>和其细菌的共生体，<em>弧菌鲵</em>，分别提出，然后介绍了允许由细菌鱿鱼轻机关的具体殖民。定植细菌衍生的发光性能和直接菌落计数检测。
Abstract
与特定的细菌被发现在动物组织1-5。这种细菌主机协会（共生）可能是有害的（致病性），有没有健身的后果（共生），或有利于（互惠）。而备受人们关注已给病原相互作用，知之甚少的进程决定的有利/共生细菌的环境重现收购。轻器官之间的海洋革兰氏阴性菌共生五鲵和夏威夷短尾鱿鱼，E. scolopes，代表着一个非常具体的互动，其中一台主机（E. scolopes）建立贯穿其一生当然6,7只有一个菌种（ 五鲵 ）的共生关系。由五，生产发光鲵在这种互动提供了一个五反掠夺利益在8,9夜间活动scolopes，而营养丰富的宿主组织提供V。鲵与受保护的利基10。在每个主机上一代，这一关系的概括，从而较可预测的过程，可以在共生发展的各个阶段的详细评估。少年鱿鱼孵化aposymbiotically（uncolonized），并在实验室中，如果前30-60分钟内收集和转移到共生的自由水，不能被殖民除实验接种6。因此，这种互动提供了一个有用的模型系统，在其中进行评估，导致从11,12环境的共生微生物的具体收购的各个步骤。
在这里，我们描述了一种方法来评估发生刚出时aposymbiotic 大肠杆菌殖民化的程度scolopes暴露（人造）海水中含有五。鲵。这个简单的实验说明接种，自然感染和恢复E.新生的光器官的细菌共生scolopes。护理提供一个一致的环境共生的发展过程中的动物，尤其是关于水质和轻线索。特征描述的共生人口的方法包括：（1）的细菌派生发光的测量，（2）直接菌落计数恢复共生。
Protocol
1。细菌菌剂的制备 0天 前两天鱿鱼接种，板13琼脂上的LBS相关的细菌株。 在25-28℃过夜孵育细菌。 第1天 接种一五，各殖民地3毫升磅玻璃文化管中等鲵株感染。准备作为备份的重复管。 第2天 （协调细菌步骤与鱿鱼步骤3.7-3.10 1.4-1.6） 1小时前接种，继代培养细菌1:80（37.5μL）…
Discussion
描述的殖民法，允许在受控的实验室环境中的一种自然的共生过程的分析。因此，它可以用来评估由不同的自然株，突变株，并根据不同的化学制度的殖民化。通常用来描述实验上的变化，以评估不同方面的共生。可以测量殖民动力学检查在第一个24小时，它可以自动检测巧合探测器已被删除，在闪烁计数器的发光。此外，一个应变，相对于其他相对定植能力，可以通过有竞争力的殖民实验测量?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢马蒂亚斯鱿鱼设施的支持和迈克尔·哈德菲尔德和Kewalo海洋实验室在现场收集的援助，并为这个协议作出贡献的Ruby和麦克福尔-艺实验室的成员在这个手稿的意见，Gyllborg。在曼德尔实验室的工作是支持由NSF IOS-0843633。
Materials
| Name of reagent | Company | Catalogue Number | Comments |
| Glass Culture Tubes, 16 mm Diameter | VWR | 47729-580 | |
| Caps for Glass Culture Tubes | Fisher | NC9807998 | |
| Visible Spectrophotometer for Determination of OD600 | Biowave | CO8000 | Any spectrophotometer capable of measuring OD600 will work. This unit can measure the OD600 of liquid directly in the glass culture tubes. Some adjustment of the inoculum calculation may be necessary depending on the instrument used. |
| GloMax 20/20 Single-Tube Luminometer | Promega | E5311 | Equivalent to the Turner BioSystems 20/20n Luminometer. Includes the microcentrifuge tube holder. |
| GloMax 20/20 Light Standard | Promega | E5341 | For luminometer calibration. |
| Refractometer, Handheld | Foster and Smith Aquatics | CD-14035 | Calibrate before each use with deionized water. Rinse after every use with deionized water to prevent salt build-up. |
| Instant Ocean (artificial seawater concentrate) | Foster & Smith Aquatics | CD-16881 | Prepare at 35 ‰ in deionized water, using the refractometer, then filter through a 0.2 μm SFCA filter. |
| Filtration Unit | Nalgene | 158-0020 | Surfactant-free cellulose acetate (SFCA) membrane, 0.2 μm. We have observed variable results with some surfactant-containing PES filters. |
| Transfer Pipettes | Fisher | 13-711-9AM | Using scissors or razor blade, cut the tip cleanly above the first ridge to increase the diameter of the pipette tip and avoid squeezing the squid hatchlings. |
| Disposable Sample Bowls (plastic tumblers) | Comet | T9S (9 oz.) | Bowls for inoculation, with upper diameter 3 ¼”, lower diameter 2 ¼”, height 3″. Bowls create a homogenous environment as they have no bottom rim, in which squid can get trapped in a low-oxygen niche. The size is optimized for 40-ml inoculum. Available at webstaurantstore.com, #619PI9. |
| Drosophila Vials | VWR | 89092-720 | Vial diameter matches the opening on the luminometer PMT. |
| 1.5 ml Microcentrifuge Tubes | ISC Bioexpress | C-3217-1CS | Tubes must fit the shape of the pestles. |
| Ethanol, 200 Proof | Fisher | BP2818-100 | |
| Pestles | Kimble Chase/Kontes | 749521-1500 | |
| Plating Beads, 5 mm diameter | Kimble Chase | 13500 5 | Prepare 5 per tube and autoclave. |
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Cite This Article
Naughton, L. M., Mandel, M. J. Colonization of Euprymna scolopes Squid by Vibrio fischeri. J. Vis. Exp. (61), e3758, doi:10.3791/3758 (2012).