Summary
Abstract
Introduction
Protocol
Representative Results
Discussion
Acknowledgements
Materials
References
Environment
这里介绍的是原位化疗的规程,这是一种最近开发的微流体装置,能够直接研究环境中的微生物行为。
微生物行为,如运动和化学(细胞改变其运动的能力,以响应化学梯度),在细菌和古生物领域广泛。在异构环境中,Chemotaxis 可产生巨大的资源获取优势。它还在共生相互作用、疾病和全球过程(如生物地球化学循环)中发挥关键作用。然而,目前的技术将化疗研究限制在实验室,并不容易应用于该领域。此处介绍的是部署原位化学测定 (ISCA) 的分步协议,该装置可直接在自然环境中对微生物化疗进行强健的检测。ISCA 是一种微流体装置,由 20 井阵列组成,可装载感兴趣的化学品。一旦部署在水环境中,化学物质会从水井中扩散出来,产生微生物通过化疗游入水井来感知和响应的浓度梯度。然后,可以对井内物质进行采样,并用于(1) 通过流式细胞学量化对特定化合物的化疗反应强度,(2) 分离和培养反应灵敏的微生物,(3) 通过分子技术描述反应群体的身份和基因组潜力。ISCA 是一个灵活的平台,可部署在水相的任何系统中,包括海洋、淡水和土壤环境。
不同的微生物利用动力和化学利用来利用零碎的营养环境,寻找宿主,或避免有害条件1,1,2,3。,3这些微生物行为反过来又会影响化学转化率4,并促进陆地、淡水和海洋生态系统2、5的共生伙伴关系。
在过去的60年里,切莫塔西在实验室条件下进行了广泛的研究。研究化疗的第一个定量方法,毛细管测定,涉及一个毛细管充满了悬浮在细菌6的悬浮液中。从管子中扩散出的化学物质会产生化学梯度,而化学细菌通过迁移到管7来响应这种梯度。自毛细管测定开发以来,目前仍然广泛使用,在日益受控的物理/化学条件下,还开发了许多其他技术来研究化学化学,最近涉及使用微流体8、9、10。,9,10
Name | Company | Catalog Number | Comments |
Acrylic glue | Evonik | 1133 | Acrifix 1S 0116 |
Acrylic sheet | McMaster-Carr | 8505K725 | Or different company |
Adhesive tape | Scotch | 3M 810 | Scotch Magic tape |
Autoclave | Systec | D-200 | Or different company |
Benchtop centrifuge | Fisher Scientific | 75002451 | Or different company |
Bungee cord | Paracord Planet | 667569184000 | Or different company |
Centrifuge tube - 2 mL | Sigma Aldrich | BR780546-500EA | Eppendorf tube |
Conical centrifuge tube - 15 mL | Fisher Scientific | 11507411 | Falcon tube |
Conical centrifuge tube - 50 mL | Fisher Scientific | 10788561 | Falcon tube |
Deployment arm | Irwin | 1964719 | Or different company |
Deployment enclosure plug | Fisher Scientific | 21-236-4 | See alternatives in manuscript |
Disposable wipers | Kimtech - Fisher Scientific | 06-666 | Kimwipes |
Flow cytometer | Beckman | C09756 | CYTOFlex |
Glutaraldehyde 25% | Sigma Aldrich | G5882 | Or different company |
Green fluorescent dye | Sigma Aldrich | S9430 | SYBR Green I - 1:10,000 final dilution |
Hydrophilic GP filter cartridge - 0.2 µm | Merck | C3235 | Sterivex filter |
In Situ Chemotaxis Assay (ISCA) | - | - | Contact corresponding authors |
Laser cutter | Epilog Laser | Fusion pro 32 | Or different company |
Luria Bertani Broth | Sigma Aldrich | L3022 | Or different company |
Marine Broth 2216 | VWR | 90004-006 | Difco |
Nylon slotted flat head screws | McMaster-Carr | 92929A243 | M 2 × 4 × 8 mm |
Pipette set | Fisher Scientific | 05-403-151 | Or different company |
Pipette tips - 1 mL | Fisher Scientific | 21-236-2A | Or different company |
Pipette tips - 20 µL | Fisher Scientific | 21-236-4 | Or different company |
Pipette tips - 200 µL | Fisher Scientific | 21-236-1 | Or different company |
Sea salt | Sigma Aldrich | S9883 | For artificial seawater |
Serological pipette - 50 mL | Sigma Aldrich | SIAL1490-100EA | Or different company |
Syringe filter - 0.02 µm | Whatman | WHA68091002 | Anatop filter |
Syringe filter - 0.2 µm | Fisher Scientific | 10695211 | Or different company |
Syringe needle 27G | Henke Sass Wolf | 4710004020 | 0.4 × 12 mm |
Syringes - 1 mL | Codau | 329650 | Insulin Luer U-100 |
Syringes - 10 mL | BD | 303134 | Or different company |
Syringes - 50 mL | BD | 15899152 | Or different company |
Tube rack - 15 mL | Thomas Scientific | 1159V80 | Or different company |
Tube rack - 50 mL | Thomas Scientific | 1159V80 | Or different company |
Uncoated High-Speed Steel General Purpose Tap | McMaster-Carr | 8305A77 | Or different company |
Vacuum filter - 0.2 µm | Merck | SCGPS05RE | Steritop filter |
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