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在西图化学分析中,以检验水生生态系统中的微生物行为

Published: May 5th, 2020

DOI:

10.3791/61062

1Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, ETH Zürich, 2Climate Change Cluster, University of Technology Sydney, 3School of Oceanography, University of Washington

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

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我们建议在实地实验之前执行第 1 节以优化结果。

1. 实验室优化

注:优化过程中描述的体积足以满足单个 ISCA(由 20 口井组成)。

  1. 感兴趣的化学品的制备
    注:在实地部署之前,通常需要在实验室条件下确定每种化疗物的最佳浓度。化学浓度场将随着与源的距离(ISCA井)而减少,这意味着环境中微生物经历的浓度将低于设备内部的浓度。在 ISCA.......

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本节介绍实验室结果,利用 ISCA 测试海洋微生物对谷氨酰胺浓度范围的化学反应,谷氨酰胺是一种已知能吸引土壤细菌的氨基酸。在实验室测试中引起最强化学反应的谷氨酰胺浓度用于在海洋环境中进行化学测定。

为了进行实验室测试,从澳大利亚悉尼沿海水域取样的海水群落通过简单的营养修正4(如步骤1.4所述)为动体细胞提供了丰富?.......

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在水生微生物的规模上,环境远非同质,其特征往往是物理/化学梯度,这些梯度结构微生物群落1,1,15。动性微生物使用行为的能力(即化疗)有助于在这些异质微环境中觅食1。直接在环境中研究化学化学具有识别重要特异性相互作用和化学偏好的潜力,这可以帮助解开特定微生物对生物地球化学过程的贡献。提出的协议将 ISCA 部署?.......

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这项研究部分由戈登和贝蒂摩尔基金会海洋微生物学倡议资助,通过向J.R.S.和R.S.提供GBMF3801赠款, 向R.S.颁发调查员奖(GBMF3783),向J.B.R颁发澳大利亚研究理事会研究金(DE160100636),西蒙斯基金会向B.S.L.(594111.)颁发奖项,西蒙斯基金会(542395)向R.S.颁发资助,作为微生物生态系统原则(PriME)合作的一部分。

....

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NameCompanyCatalog NumberComments
Acrylic glueEvonik1133Acrifix 1S 0116
Acrylic sheetMcMaster-Carr8505K725Or different company
Adhesive tapeScotch3M 810Scotch Magic tape
AutoclaveSystecD-200Or different company
Benchtop centrifugeFisher Scientific75002451Or different company
Bungee cordParacord Planet667569184000Or different company
Centrifuge tube - 2 mLSigma AldrichBR780546-500EAEppendorf tube
Conical centrifuge tube - 15 mLFisher Scientific11507411Falcon tube
Conical centrifuge tube - 50 mLFisher Scientific10788561Falcon tube
Deployment armIrwin1964719Or different company
Deployment enclosure plugFisher Scientific21-236-4See alternatives in manuscript
Disposable wipersKimtech - Fisher Scientific06-666Kimwipes
Flow cytometerBeckmanC09756CYTOFlex
Glutaraldehyde 25%Sigma AldrichG5882Or different company
Green fluorescent dyeSigma AldrichS9430SYBR Green I - 1:10,000 final dilution
Hydrophilic GP filter cartridge - 0.2 µmMerckC3235Sterivex filter
In Situ Chemotaxis Assay (ISCA)--Contact corresponding authors
Laser cutterEpilog LaserFusion pro 32Or different company
Luria Bertani BrothSigma AldrichL3022Or different company
Marine Broth 2216VWR90004-006Difco
Nylon slotted flat head screwsMcMaster-Carr92929A243M 2 × 4 × 8 mm
Pipette setFisher Scientific05-403-151Or different company
Pipette tips - 1 mLFisher Scientific21-236-2AOr different company
Pipette tips - 20 µLFisher Scientific21-236-4Or different company
Pipette tips - 200 µLFisher Scientific21-236-1Or different company
Sea saltSigma AldrichS9883For artificial seawater
Serological pipette - 50 mLSigma AldrichSIAL1490-100EAOr different company
Syringe filter - 0.02 µmWhatmanWHA68091002Anatop filter
Syringe filter - 0.2 µmFisher Scientific10695211Or different company
Syringe needle 27GHenke Sass Wolf47100040200.4 × 12 mm
Syringes - 1 mLCodau329650Insulin Luer U-100
Syringes - 10 mLBD303134Or different company
Syringes - 50 mLBD15899152Or different company
Tube rack - 15 mLThomas Scientific1159V80Or different company
Tube rack - 50 mLThomas Scientific1159V80Or different company
Uncoated High-Speed Steel General Purpose TapMcMaster-Carr8305A77Or different company
Vacuum filter - 0.2 µmMerckSCGPS05RESteritop filter

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