Quantification of Respiratory Activity in Biofilms
生物膜呼吸活性的量化
发布: 2015年09月20日第5卷第18期 DOI: 10.21769/BioProtoc.1591 浏览次数: 10154
评审: Maria SinetovaAlexander B. WestbyeEsteban Paredes-Osses
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参见作者原研究论文
The authors used this protocol in:
Nov 2014
Abstract
Bacteria live mostly as biofilms, not as planktonic cell populations. Bacterial cells living as biofilms are known to be in different physiological status. Persister cells are one of such physiological conditions and they are recognized as to be a stochastically produced sub-population of non-growing bacterial cells. The following protocol describes a method to determine the respiratory activity of cells within biofilms.
Materials and Reagents
- Bacterial culture [Pseudomonas aeruginosa (P. aeruginosa) PA14]. This method has also been performed for Escherichia coli (BW25113)
- LB broth Lennox (BD, catalog number: 240230 )
- CorningTM cellgroTM Ciprofloxacin Hydrochloride (Thermo Fisher Scientific, catalog number: 61-277-RF )
- BacLightTM RedoxSensorTM CTC Vitality Kit (Thermo Fisher Scientific, catalog number: B34956 )
- SYTO® 40 Blue Fluorescent Nucleic Acid Stain-5 mM Solution in DMSO (Thermo Fisher Scientific, catalog number: S11351 )
- Sodium chloride (VWR International, BDH®, catalog number: 0241-VBD )
- Aluminum Foil
- Spooled Masterflex peroxide-cured silicone tubing, L/S 14, 250 ft. (Cole-Parmer, catalog number: UX-96407-14 )
- Masterflex Norprene tubing (A60 G), L/S 13, 50 ft. (Cole-Parmer, catalog number: UX-06404-13 )
- PVDF barbed Y connector, 3/8" ID, 1/4", 2-3/8", 1-3/4"; Pack Of 10 (Cole-Parmer, catalog number: WU-30703-93 )
- Barbed fittings, Straight Connector, Clear PP,1/16" ID, 1/32", 25/32", 1/4" (Cole-Parmer, catalog number: WU-30506-00 )
- Barbed fittings, Reducing Connector, Clear PP,1/8" x 1/16" ID, 3/32", 13/16", 1/8" (Cole-Parmer, catalog number: WU-30506-06 )
- Barbed fittings, T connector, Kynar, 1/4" ID, 1/8", 1-15/16", 1-5/16"; 10/pack (Cole-Parmer, catalog number: WU-30703-75 )
- Micropipettes (P1000, P200, P20)
- Syringe 10 ml
- Syringe needle
- Glass microscope slides (25 x 75 x 1 mm)
- Glass microscope coverslips (60 x 24 mm; no 2)
- Saline (see Recipes)
Equipment
- Peristaltic pump (Cole-Parmer, catalog number: EW-07553-80) with an 8 channel, 6 rollers, 3-stop Ismatec minicartridge pump head (Cole-Parmer, catalog number: EW-78002-50 )
- Inoculation ports (VWR® Sleeve Stoppers) (VWR International, catalog number: 89097-534 )
- GeneMate Incubated Shakers (BioExpress, catalog number: H-2000-M )
- Nalgene® Carboys with Handles, Polypropylene, Thermo Scientific (VWR International, catalog number: 16101-084 )
- Nalgene® Top WorksTM Aseptic Closure System, Silicone, for Bottles and Carboys, Thermo Scientific (VWR International, catalog number: 2135-8303 )
- Acro® 50 Vent Filters, Pall Laboratory (VWR International, catalog number: 28143-616 )
- Anodized Transmission flow cell reactors (Biosurface technologies, catalog number: FC 81-Al )
- Confocal Scanning Microscope and Analysis software (e. g. Leica Confocal SP5 Imaging system and Software)
Software
- COMSTAT software (http://www.comstat.dk)
- Intensity Luminance V1 software (http://bingweb.binghamton.edu/~scraver/IL.html)
- ImageJ software (http://imagej.nih.gov/ij/)
- Leica LAS AF software (Leica)
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Marques, C. N. H. and Craver, S. A. (2015). Quantification of Respiratory Activity in Biofilms. Bio-protocol 5(18): e1591. DOI: 10.21769/BioProtoc.1591.
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分类
微生物学 > 微生物生物膜 > 生物膜培养
微生物学 > 微生物细胞生物学 > 细胞染色
微生物学 > 微生物生理学 > 呼吸
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