微生物菌落生物膜的石蜡嵌入和薄切片显微分析
Summary
我们描述了微生物菌落生物膜的固定、石蜡嵌入和薄切片技术。在制备的样品中, 生物膜子结构和记者的表达模式可以通过显微技术进行可视化。
Abstract
石蜡切片是一种在真核细胞中广泛建立的技术。在这里, 我们提供了一种方法, 以固定, 嵌入和切片的完整的微生物群生物膜使用灌注石蜡。为了适应这种方法在菌落生物膜上的使用, 我们开发了技术, 以保持每个样品在其生长基质和层压它与琼脂层叠, 并添加赖氨酸的固定液。这些优化改进了果皮微形态特性的样本保留和保存。以这种方式制备的样品可以通过光、荧光和透射电镜进行薄切片和成像。我们已将此技术应用于铜绿假单胞菌、假单胞菌 synxantha、枯草芽孢杆菌和霍乱弧菌的菌落生物膜。该方法所产生的样品中可见的高度细节, 加上报告应变工程或特定染料的使用, 可为微生物群落的生理和发育提供令人振奋的见解。
Introduction
大多数微生物都有能力形成生物膜, 细胞群落由自制的基质聚集在一起。生物膜可以生长在许多类型的物理设置, 与各种制度的营养和基质供应。特定的生物膜形成方法往往产生可重复的多细胞结构, 并观察到在群落或宏观层面上 phylogenetically 不同物种的共同结构。当微生物在大气层中作为固体介质的菌落生长时, 宏观形态传达有关矩阵生产能力的信息, 并且经常与其他特性相关, 1,2,3。微生物菌落的内部结构也可以提供有关生物膜特异化学和生理学的线索, 但很难描述。最近, cryoembedding 和 cryosectioning 技术在细菌菌落中的应用已使特定功能的成像和可视化达到空前的分辨率4,5,6。然而, 与动物组织的研究表明, 石蜡嵌入提供了卓越的保存形态, 当与 cryoembedding 7相比, 并已用于可视化的细菌在组织8,9。因此, 我们制定了一个固定, 石蜡嵌入和薄切片微生物菌落生物膜的协议。在这里, 我们将描述铜绿假单胞菌 PA14 菌落-生物膜薄部分10,11的制备, 但我们也成功地将此技术应用于细菌形成的生物膜铜绿假单胞菌 synxantha, 枯草芽孢杆菌,和霍乱弧菌12。
石蜡嵌入和薄切片生物膜的过程遵循一个简单的逻辑。首先, 生物膜被包裹在一层琼脂中, 以保持加工过程中的形态学。第二, 包裹生物膜被浸没在一个固定剂中, 以交联大分子并保存形貌。然后用酒精脱水, 用更非极性溶剂清除, 然后用液体石蜡渗透。一旦渗入, 样品被嵌入到蜡块中切片。节被剪切, 装在幻灯片上, 然后水化以返回到更纯的状态。从这一点上, 它们可以被染色或覆盖在安装介质中进行微观分析。
本协议生产适合于组织学分析的微生物生物膜薄切片。当使用这种方法制备的薄切片被光镜成像时, 菌落生物膜子结构可见。生物膜也可以在含有特定于个别特征的荧光污渍的介质上生长, 或者在安装前立即在补液步骤 (步骤 9.5-9.6) 上染色。最后, 微生物可以被设计成以一种本构或调控的方式生产荧光蛋白, 允许就地报告这些社区内的细胞分布或基因表达。利用这些方法确定了菌落的生物膜深度、细胞分布、基质分布、生长形态和时空基因表达。
Protocol
1. 铜绿假单胞菌菌落生物膜的生长 中双层板的制备 准备一10克/升胰蛋白胨, 10 克/升琼脂 (参见材料表) 溶液在去离子水。 20分钟的蒸压釜, 在水浴中冷却至50-60 摄氏度。 将45毫升的琼脂-胰蛋白胨溶液倒入100毫米 x 100 毫米方盘 (参见材料表), 使用50毫升锥形管。允许琼脂凝固 (~ 20-30 分钟)。在第一层上倒入第二个, 15 毫升的层。让一夜之间凝固, …
Representative Results
该方法产生的生物膜薄切片, 其中明显的形态学特征和区域的基因表达可以成像的 DIC, 荧光显微镜, 和 TEM。虽然 DIC 成像使用一个40X 的油浸泡目标可以足以显示一些形态学特征 (图 2E), 我们发现, 荧光显微镜的菌株设计的组成性快速荧光蛋白提供了增强示例中单元格分布的可视化 (图 2D)。可以将单个剖面的图像缝合在一起, 生成?…
Discussion
石蜡嵌入和薄切片组织标本是一种经典的组织学技术, 使显微形态学结构的成像和通常用于真核组织, 并已成功地应用于微生物样本8 ,9。虽然 cryoembedding 允许强保留内源和免疫荧光信号, 石蜡嵌入通常是可取的, 因为它提供更好的保存形态学16。在将这种方法应用于微生物生物膜时, 我们关注的是我们系统特有的特性。组织的细胞和基质?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了 NSF 职业奖1553023和 NIH/NIAID 奖 R01AI103369 的支持。
Materials
| 5 3/4" Pasteur pipette | Fisher Scientific | 13-678-6A | Purchased from univeristy biostores |
| Agar | Teknova | A7777 | |
| Buchner Aspirator (Vacuum) Flask | Pyrex | 5340 | Purchased from univeristy biostores |
| Chemically-resistant Marking Pen | VWR | 103051-182 | Manufacturer: Leica |
| Clear Fingernail Polish | ******** | ******** | Store bought |
| Congo Red Indicator Grade | VWR | AAAB24310-14 | Manufacturer: Alfa Aesar |
| Coomassie Blue | VWR | EM-3340 | Manufacturer: EMD Millipore |
| TRIS-buffered Mounting Medium (w/ DAPI) | Fisher Scientific | 50 247 04 | Manufacturer: Electron Microscopy Sciences |
| Embedding Mold | ******** | ******** | 3D printed in-house |
| Embedding Mold (commercial) | Electron Microscopy Sciences | 70182 | |
| Ethanol 200P | Decon Labs, Inc. | 2701 | Purchased from univeristy biostores |
| Fine-tipped Brush | ******** | ******** | Store bought, paint brush |
| Glass Coverslips 60x22mm | Fisher Scientific | 12-519-21C | |
| Glass Rehydration Mailer | Ted Pella | 21043 | 20 slide mailer |
| Histoclear-II, orange oil-based clearing agent | Fisher Scientific | 50 899 90150 | Manufacturer: National Diagnostics |
| Histosette, Embedding Casette | Fisher Scientific | 15 182 701A | |
| L-lysine hydrochloride | Fisher Scientific | BP386 100 | |
| Low Profile Microtome Blades | Fisher Scientific | 22 210 048 | Manufacturer: Sturkey |
| Micropipette | VWR | 89080-004 | Promo-pack |
| Micropipette Tips | See comments section | See comments section | p10 (Fisher Scientific, 02 707 469), p200 (VWR, 89079-474), p1250 (VWR, 89079-486) |
| Microtome | Fisher Scientific | 905200U/00016050 | Model: HM355S, Manufacturer: Microm, NON-CATALOG, Vendor Catalog # 905200U/00016050 |
| Formaldehyde, 37% Aqueous (Formalin) | Ricca Chemical | RSOF0010-500A | |
| Paraplast Xtra (paraffin wax) | VWR | 15159-486 | Manufacturer: McCormick Scientific |
| Petri Dishes Square 100x100x15mm | Laboratory Disposable Products | D210-16 | |
| Potassium chloride | EMD Chemicals | PX1405-1 | Component of phosphate buffered saline, prepared in-house |
| Potassium phosphate | Fisher Scientific | P380-500 | Component of phosphate buffered saline, prepared in-house |
| Razor Blades | VWR | 55411-050 | Purchased from univeristy biostores |
| Slide Warmer | Fisher Scientific | NC0865259 | NON-CATALOG, Vendor Catalog # 12857D |
| Sodium chloride | VWR | 0241-1KG | Component of phosphate buffered saline, prepared in-house |
| Sodium phosphate | VWR | BDH9296.500 | ,Component of phosphate buffered saline, prepared in-house |
| Suprafrost Histology Slides | Fisher Scientific | 12-544-2 | |
| Tissue Flotation Water Bath | Fisher Scientific | NC0815797 | Manufacturer: Ted Pella, Vendor Catalog # 28156-B |
| Automatic Tissue Processor | Fisher Scientific | 813160U/Q#00009061 | Model: STP120 Tissue Processor |
| Tryptone | Teknova | T9012 | |
| Yeast extract | Teknova | Y9010 |
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Citer Cet Article
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