Bacterial Fluorescent-dextran Diffusion Assay
细菌荧光右旋糖酐扩散分析
发布: 2014年07月20日第4卷第14期 DOI: 10.21769/BioProtoc.1191 浏览次数: 9895
评审: Anonymous reviewer(s)
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The authors used this protocol in:
Aug 2013
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Abstract
Antimicrobial peptides are known to disrupt bacterial membranes allowing solutes to flow across the membrane in an unregulated manner resulting in death of the organism. Disrupting the bacterial membrane would thus perturb the cells osmotic balance resulting in an initial influx of the external aqueous buffer. We have designed an assay to investigate how antimicrobial peptide concentration affects the ability of fluorescently labelled dextran moieties of differing molecular weight and hydrodynamic radii to cross membranes of viable bacteria. This assay was used to show that diffusion of low and high molecular weight dextrans into bacteria was a function of antimicrobial peptide concentration (Sani et al., 2013).
Antimicrobial peptides (抗菌肽)
Fluorescent dye (荧光染料)
Cell osmotic balance (细胞渗透平衡)
Membrane diffusion (膜扩散)
Materials and Reagents
- Bacteria of interest [e.g. Staphylococcus aureus (S. aureus)] (Sani et al., 2013)
- Rhodamine-dextran (RD) of different molecular weights [e.g. 40 kDa RD molecular weight (RD-40) (Sigma-Aldrich, catalog number: 42874 )]
- Fluorescein-dextran (FD) of different molecular weights [e.g. 4.4 kDa FD (FD-4) (Sigma-Aldrich, catalog number: 46944 )]
- LIVE/DEAD® BacLightTM Bacterial Viability and Counting Kit (Life Technologies, catalog number: L34856 )
- Dulbecco’s Phosphate Buffered Saline (Dulbecco’s PBS) (Sigma-Aldrich, catalog number: D8537 )
- Peptide of interest (e.g. Maculatin 1.1) (Sani et al., 2013)
- Bacterial growth media [i.e., particular for the bacteria of interest e.g. S. aureus was grown as a batch culture in Luria broth (Thermo Fisher Scientific) (see Recipes)] (Sani et al., 2013)
Equipment
- Flow cytometer (e.g. Beckman Coulter, model: Quanta SC-MPL)
- 96 well plates (flat bottomed) (Thermo Fisher Scientific, Nunc®, catalog number: 12565210 )
- Transfer pipette
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
O’Brien-Simpson, N. M., Pantarat, N., Walsh, K. A., Reynolds, E. C., Sani, M. and Separovic, F. (2014). Bacterial Fluorescent-dextran Diffusion Assay. Bio-protocol 4(14): e1191. DOI: 10.21769/BioProtoc.1191.
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分类
微生物学 > 抗微生物试验 > 抗菌肽试验
微生物学 > 微生物细胞生物学 > 细胞活力
生物化学 > 糖类 > 多糖
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