这篇文章提出了不断增长的生物膜的原位飞行时间二次离子质谱的研究化学的映射在其水化状态下,由微流控反应器,在液体真空接口分析系统启用的方法。希瓦氏菌 oneidensis先生-1 与绿色荧光蛋白作为一种模型。
细菌生物被膜表面相关的社区大大研究了解他们自身产生的胞外聚合物 (EPS) 和在环境微生物学中的作用。本研究概述了培养生物膜附着到对系统进行分析在液体真空接口 (萨尔) 和飞行时间二次离子质谱 (TOF-SIMS) 实现原位化学映射的生活生物膜的方法。这样做是通过培养细菌外和内萨尔维通道与我们专门的安装程序,并通过光学成像技术来检测生物膜的存在和 TOF-SIMS 分析前的厚度。我们的研究结果表明希瓦氏菌生物膜的特征峰在其自然的水合状态,突出其本地化的水群集环境中,以及 EPS 片断,它们是截然不同的同一生物膜脱水状态。这些结果表明基于真空化学成像仪原位生物膜成像允许的萨尔的突破能力。
细菌生物被膜表面相关社区给细菌提供生存变不利的物理和机械刺激,其中细胞都能够重视,并在许多可能的环境中生存的防御随着时间推移逐渐形成了。1,2生物膜大大调查和在生物医学、 生物医学工程、 农业和工业研究和发展等很多领域有应用。1,2理解这些复杂的微生物群落,包括他们自身产生的胞外聚合物 (EPS) 和他们的当地水-群集环境中,化学图谱至关重要获得准确和详细其生物活动的描述。2
生物膜存在,但在高度水化状态的成长。这带来一个巨大的挑战,在使用基于真空的表面分析技术,如飞行时间二次离子质谱 (TOF-SIMS) 由于研究挥发性液体在真空中的困难。因此,基于真空的表面分析技术已几乎完全限于研究生物膜的样本,只是他们干的状态。然而,研究其干州生物膜抑制准确调查其真实的生物微环境。它常常引起 EPS 完整性和生物膜形态,比较干膜质谱结果到原位液体研究后证明了剧变。3,4这篇文章提出了一个解决方案利用我们的系统用于分析在液体真空接口 (萨尔),5,6微流控反应器研究生物膜内他们自然的水合状态,包含在其薄硅氮化 (SiN) 膜在微通道的聚二甲基硅氧烷 (精原细胞瘤),从而同时仍保持在真空内的液体矩阵的结构完整性提供直接访问二次离子探针光束下的液态分庭。7,8
S.oneidensis先生-1 突变表达绿色荧光蛋白 (GFP) 被选为模式生物的由于其代谢的多功能性和共同使用的环境和应用微生物学,基于此生物膜过程图很大程度上金属还原和胞外电子转移的独特能力。9,10,11此外,绿色荧光蛋白的存在允许容易连续生物膜-厚度监测通过荧光显微镜、 荧光素异硫氰酸酯 (FITC) 筛选器使用。我们以往的研究表明倾向于使用在原位荧光成像技术对生物膜的生长厚度达 100 微米的罪窗口的附件此细菌的证据。4,12虽然本文将只讨论通过荧光显微镜观察生物被膜的存在的确认,萨尔维是兼容其他光学超分辨成像方法荧光成像 (即结构照明显微镜 (SIM)9) 和共焦激光扫描显微镜 (CLSM) 成像4)。光学成像可以作为衡量生物膜厚度,并获取三维图像的生物膜的形状看来,确认其厚度和其附件罪窗口。9虽然 GFP 用于 SIMS 分析, S.oneidensis没有绿色荧光蛋白用作这唯一所需测量的光学密度的生长曲线,并不需要作任何荧光成像。一般来说的区别 GFP 标记和未加标签的造林树种生长曲线是微不足道。此外,虽然本议定书使用美国 oneidensis先生 1 GFP 作为模式生物来描述过程,此过程旨在为任何可能需要在萨尔内培养的细菌菌株。虽然鉴于菌株所需的知识,一些生长条件,如时间、 温度和氧气环境可能需要修改,以适应的菌株,用于。对于生长介质,此过程使用”纳米”中等、 胰酪胨大豆肉汤 (TSB) 葡萄糖,而胰酪胨大豆琼脂 (TSA) 葡萄糖不培养。”纳米”培养基的组成已专门制定了为增长和监测膜扩展和美国 oneidensis出现初具规模的小电线及培养基成分质已在以往的研究内设立。13,14
我们以前的协议,在原位上液体 TOF-SIMS 阐释了萨尔维已为蛋白质固定化和罪恶,以及 TOF-SIMS 分析和数据减少详细的议定书的附件提供的好处。12 ,而不是重申数据减少步骤,本文将有助于集中精力建立和培养生物膜内我们萨尔维微通道,以及成像的步骤来检测生物膜的存在和厚度事先的独特方法TOF-SIMS 分析。而生物膜有以前仅限于只干燥样品内室基于真空的表面分析技术,详细的 EPS 和生物膜化学映射的活生物膜现在可原位由于这一新功能。
后接种日志阶段,它是重要测试数天和在其中生物膜应成长之前它是健康和成像,够厚在第 3.1 步中所述的温度。此过程具体包括培养 S.oneidensis MR1 生物膜在室温;然而不同的室内温度可以影响率的增长。因此,使用光学成像来了解生物膜是否已准备好之前 TOF-SIMS 分析至关重要。同样,不同株细菌需要不同生长条件和长度来实现理想的厚度进行分析。图 1b 中的生长曲线描绘了这种细菌要 12-32 h 第 2…
The authors have nothing to disclose.
我们非常感谢太平洋西北国家实验室 (单比) 地球和生物科学 (EBD) 特派团种子实验室指导研究与发展 (LDRD) 基金的支持。通过 W.R.威利环境分子科学实验室 (其) 一般用户建议提供了工具的访问。其是由办公室生物和环境研究 (BER) 在太平洋西北国家实验室主办全国科学用户设施。作者感谢丁博士曌校对手稿和提供有用的反馈。太平洋西北国家实验室是美国能源部下合同 DE-AC05-76RL01830 由巴特尔推行。
ToF-SIMS | IONTOF | TOF.SIMS 5 | Resolution:>10,000 m/Δm for mass resolution;>4,000 m/Δm for high spatial resolution |
System for Analysis at the Liquid Vacuum Interface (SALVI) | Pacific Northwest National Laboratory | N/A | SALVI is a unique, self-contained, portable analytical tool that, for the first time, enables vacuum based scientific instruments such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) to analyze liquid surfaces in their natural state at the molecular level. |
-80°C Freezer | New Brunswick Scientific | N/A | U410 Premium Energy Efficient Ultra-Low Temperature Freezer |
4°C Refrigerator | BioCold Scientific | N/A | COLDBOX1 |
Orbital Shaker | New Brunswick Scientific | N/A | Innova 4900 Multi-Tier Environmental Shaker, set at 30 degrees Celsius for serum bottle and flask culturing, set at 150rpm. |
Syringe Pump | Cole-Parmer | EW-74905-02 | Cole-Parmer Syringe Pump, Infusion Only, Touchscreen Control 74905-02, used for injecting liquid into the tubing system and SALVI at a constant flowrate. |
Incubator | Barnstead International | LT1465X3 | Lab-Line incubator, set at 30 degrees Celsius for plate culturing. |
Autoclave | Getinge | 533LS | Used to sterilize PEEK fittings, tubing systems, serum vials, and medium. Model 533LS Vacuum Steam Sterilizer |
Spectrophotometer | Thermo Fisher Scientific | 4001-000 | GENESYS 20 spectrophotometer for OD600 readings of cuvettes for growth curves. |
Biological Safety Cabinet | Thermo Fisher Scientific | 1385 | 1300 Series AZ Biological Safety Cabinet |
Fluorescence Microscope | Nikon | N/A | Nikon OPTIPHOT-2 fluorescence microscope with camera and super high pressure mercury lamp power supply. |
pH Meter | Mettler Toledo | 51302803 | Used to test the pH of the “nanowires” medium after finished and before autoclaving. |
PEEK Union | Valco | ZU1TPK | For connecting the inlet and outlet of SALVI, the syringe to the tubing system, and the inlet of the SALVI to the drip chamber of the tubing system. |
5 Axes Sample Stage | IONTOF | N/A | Stage is self-made for mounting SALVI in ToF-SIMS. |
Barnstead Nanopure Water Purification System | Thermo Fisher Scientific | D11921 | ROpure LP Reverse Osmosis filtration module (D2716) |
Pipette | Thermo Fisher Scientific | 21-377-821 | Range: 100 to 1,000 µL. |
Pipette Tip | Neptune | 2112.96.BS | 1,000 µL pipette tips |
Razor Blade Handle | Stanley | N/A | Stanley Bostitch Razor Blade Scraper with 5 Single-Edge Blades, used for cutting PTFE tubing |
Syringe | BD | 309659 | 1 mL |
Syringe | BD | 309657 | 3 mL |
Syringe | BD | 309646 | 5 mL; Used for making the drip chamber |
Syringe | BD | 309604 | 10 mL |
Syringe | BD | 302830 | 20 mL |
Disposable Pipette | Thermo Fisher Scientific | 13-678-11 | 25 mL Fisherbrand™ Sterile Polystyrene Disposable Serological Pipets with Magnifier Stripe, for filling serum bottles. |
Electric Pipette Filler | Pipet-aid | P-57260 | Vacuum pressure electric serological pipette filler |
Serum Bottle | Sigma | 33109-U | Holds approximately 69 mL of liquid for culture growth, optimum for use of 20mL culture per bottle. |
Anaerobic Culture Tube | VWR | 89167-178 | Anaerobic Tubes, 18 x 150 mm, Supplied with 20 mm Blue Butyl Rubber Stopper and Aluminum Seal. |
Rubber Stopper | Sigma | 27235-U | Silicone stopper, used for sealing serum bottles and for creating the tubing system/drip chamber. |
Aluminum Crimp Seal (without septum) | Sigma | 27227-U | Aluminum seal for top of serum bottle for use with serum bottle crimper. |
Serum Bottle Aluminum Seal Crimper | Wheaton | 224307 | 30 mm crimper with standard seal. |
PTFE Tubing | Supelco | 58697-U | 1.58 mm OD x 0.5 mm ID 50 ft. PTFE Teflon tubing, used for creating the tubing system. |
Disposable Cuvettes | GMBH | 759085D | 1.5 Ml for use with spectrophotometer. |
Needle | BD | 303015 | 22G; used for serum bottle injection. |
Needle | BD | 305120 | 23G; used for punching-through rubber stopper to create drip tubing system. |
Shewanella oneidensis MR-1 with GFP | N/A | N/A | Matthysse AG, Stretton S, Dandie C, McClure NC, & Goodman AE (1996) Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli. FEMS Microbiol Lett 145(1):87-94. |
Ethanol | Thermo Fisher Scientific | S25310A | 95% Denatured |
TSA | BD | 212305 | Tryptic soy agar for culturing the model organism (S. oneidensis) used in this protocol |
PIPES Buffer | Sigma | P-1851 | Used for “nanowires” medium {Hill, E.A. 2007} |
Sodium Hydroxide | Sigma | S-5881 | Used for “nanowires” medium {Hill, E.A. 2007} |
Ammonium Chloride | Sigma | A-5666 | Used for “nanowires” medium {Hill, E.A. 2007} |
Potassium Chloride | Sigma | P-4504 | Used for “nanowires” medium {Hill, E.A. 2007} |
Sodium Phosphate Monobasic | Sigma | S-9638 | Used for “nanowires” medium {Hill, E.A. 2007} |
Sodium Chloride | Thermo Fisher Scientific | S271-3 | Used for “nanowires” medium, and used to make mineral solution used for “nanowires” medium {Hill, E.A. 2007} |
Sodium lactate | Sigma | L-1375 | 60%(w/w) syrup @ 98% pure, d=1.3 g/mL, 7M, used for “nanowires” medium {Hill, E.A. 2007} |
Sodium Bicarbonate | Sigma | S-5761 | Used to make ferric NTA solution, used for “nanowires” medium {Hill, E.A. 2007} |
Nitrilotriacetic Acid Trisodium Salt | Sigma | N-0253 | Used to make ferric NTA solution, used for “nanowires” medium {Hill, E.A. 2007} |
Iron (III) Chloride | Sigma | 451649 | Used to make ferric NTA solution, used for “nanowires” medium {Hill, E.A. 2007} |
Magnesium Sulfate | Sigma | 208094 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Manganese (II) Sulfate Monohydrate | Sigma | M-7634 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Iron(II) Sulfate Heptahydrate | Sigma | 215422 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Calcium Chloride Dihydrate | Sigma | 223506 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Cobalt(II) Chloride | Sigma | 60818 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Zinc Chloride | Sigma | 229997 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Copper(II) Sulfate Pentahydrate | Sigma | C-8027 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Aluminum Potassium Sulfate Dodecahydrate | Sigma | 237086 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Boric Acid | Sigma | B-6768 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Sodium Molybdate Dihydrate | Sigma | 331058 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Nickel(II) Chloride | Sigma | 339350 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
Sodium Tungstate Dihydrate | Sigma | 14304 | Used to make minerals solution, used for “nanowires” medium {Hill, E.A. 2007} |
D-Biotin | Sigma | 47868 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
Folic Acid | Sigma | F-7876 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
Pyridoxine Hydrochloride | Sigma | P-9755 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
Riboflavin (B2) | Sigma | 47861 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
Thiamine Hydrochloride | Sigma | T-4625 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
Nicotinic Acid | Sigma | N4126 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
D-Pantothenic Acid Hemicalcium Salt | Sigma | 21210 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
Vitamin B12 | Sigma | V-2876 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
4-Aminobenzoic Acid | Sigma | A-9878 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |
Thioctic Acid | Sigma | T-1395 | Used to make vitamin solution, used for “nanowires” medium {Hill, E.A. 2007} |