原位萨尔和 TOF-SIMS希瓦氏菌 oneidensis MR1 生物膜的表征

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1.制备的材料 介质油管制备 （一个需要每个生物膜文化和三个需要每生长曲线） 的血清瓶 注： 引进、 任何成长所述中等适宜提供所需的感兴趣的菌株营养可以用于此过程;在这种情况下，" 纳米线 " 媒体和 TSB 没有葡萄糖培养基被用于 美国 oneidensis 先生 1 GFP 的增长。 13 存款 20 毫升的增长中进了一个 70 毫升血清瓶，盖塞子和卷…

Representative Results

这些代表性的结果，足以说明如何可以识别和解释，附生物膜的化学特征，如通过 TOF-SIMS 获得。后密谋从 TOF-SIMS 数据采集，在程序部分，简要地强调质谱峰值识别应进行将标识分配给每个各自的 m/z 值。这可以通过广泛的文献回顾对质谱在细菌和预计中研究了等各种水团簇、 脂肪酸和蛋白质片段的细菌可能存在的特定化学片段。16,…

Discussion

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…

Materials

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}