原子力显微镜成像的细菌固定化
1Biological and Nanoscale Systems Group, Biosciences Division,Oak Ridge National Laboratory, 2Department of Biochemistry and Cellular and Molecular Biology,University of Tennessee , 3Department of Surgery,Eastern Virginia Medical School, 4Center for Nanophase Materials Sciences Division,Oak Ridge National Laboratory
Summary
现场革兰氏阴性和革兰氏阳性菌，可固定在明胶包覆云母和利用原子力显微镜（AFM）在液体中成像。
Abstract
AFM是一个高分辨率（纳米尺度）的成像工具，机械探头表面。它具有图像细胞和生物分子的能力，在液体环境中，无需化学处理的样品。为了实现这一目标，必须充分坚持样品安装表面，以防止去除扫描原子力显微镜悬臂的针尖施加的力量。在许多情况下，成功的成像取决于样品固定在安装表面。理想情况下，固定应微创样品等的代谢过程和功能属性不受损害。猪（猪）明胶涂层新鲜剥离的云母表面带负电荷的细菌可以固定在表面上，并在液体中原子力显微镜成像。明胶包覆云母上的细菌细胞固定化是最有可能是由于带负电荷的细菌和带正电荷的明胶之间的静电相互作用。有几个因素可以干扰细菌固定化，包括在其中的细菌悬浮液，明胶包覆云母，菌株和成像在其中的细菌培养基的表面特性的细菌培养时间化学成分。总体而言，使用明胶包覆云母成像微生物细胞被普遍适用。
Protocol
1。云母的准备： 削减云母的大小要适合AFM显微镜（约22 × 30毫米）的剪刀（电子显微镜学）。 顺劈斩双方云母，一般用胶带去除的外层，直到顺利不间断层保持。 2。明胶溶液的制备： 实验室瓶100毫升的蒸馏水。 一瓶在微波加热，直到水开始沸腾。 （如果不可用微波炉，水可以在一个热点板块的烧杯加热。） 称取0.5克，明胶（Sigma?…
Discussion
许多因素可能会影响微生物细胞固定的原子力显微镜和成像。明胶，用于涂料，云母是重要的。商业明胶是孤立的，从脊椎动物，包括鱼，牛，猪的数量。的起源和处理方法，确定为细菌固定化明胶的适用性。许多来源和类型的明胶固定细菌的有效性进行评估[1]。两个最有效的明胶被发现SIGMA的G – 6144和G – 2625。从猪（猪），这些明胶被认为是低布鲁姆和中等布鲁姆分别。明胶处理过的基材和特定?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是，美国能源部生物和环境研究办公室，并授予来自弗吉尼亚州的联邦卫生研究委员会的资金赞助。美国能源部橡树岭国家实验室管理有限责任公司，UT斯达康，巴特尔，根据合同号DE – AC05 – 00OR22725。
Materials
| Name | Company | Catalogue number |
| Gelatin | Sigma, St. Louis, MO | G6144, G2625 or G2500 |
| PicoPlus Atomic Force Microscope | Agilent Technologies, Tempe, AZ | |
| AFM cantilevers | Veeco, Santa Barbara, CA | MLCT-AUHW |
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Bacterial ImmobilizationAtomic Force MicroscopyHigh-resolution ImagingImaging CellsBiomoleculesLiquid EnvironmentScanning AFM Cantilever TipSample ImmobilizationMinimally Invasive ImmobilizationPorcine Gelatin CoatingNegative BacteriaElectrostatic InteractionFactors Affecting ImmobilizationMicrobial Cell Imaging
Cite This Article
Allison, D. P., Sullivan, C. J., Mortensen, N. P., Retterer, S. T., Doktycz, M. Bacterial Immobilization for Imaging by Atomic Force Microscopy. J. Vis. Exp. (54), e2880, doi:10.3791/2880 (2011).