从绿脓杆菌生物膜和浮游种群中分离持留细胞

Cláudia N. H. Marques

doi:10.21769/BioProtoc.1590

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Peer-reviewed

Isolation of Persister Cells from Biofilm and Planktonic Populations of Pseudomonas aeruginosa

从绿脓杆菌生物膜和浮游种群中分离持留细胞

Cláudia N. H. Marques email

发布: 2015年09月20日第5卷第18期 DOI: 10.21769/BioProtoc.1590 浏览次数: 12440

评审: Maria SinetovaAlexander B. WestbyeEsteban Paredes-Osses

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Cover of Applied and Environmental Microbiology, featuring study using the protocol.

Nov 2014

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Abstract

Persister cells are a stochastically produced sub-population of non-growing bacterial cells. Recently these cells have been more widely studied due to the recognition that they are tolerant to antimicrobials and thus, play a major role in the resilience of bacterial populations to antimicrobials, particularly in chronic biofilm infections. The following protocol describes the isolation/selection of persister cell sub-populations of Pseudomonas aeruginosa present in biofilms (sessile) and planktonic populations (free-living).

Keywords: Persister cells (耐药株细胞)

Pseudomonas aeruginossa (aeruginossa假单胞菌)

Biofilms (生物膜)

Tube reactors (管式反应器)

Planktonic populations (浮游种群)

Materials and Reagents

Bacterial culture [Pseudomonas aeruginosa PA14]. This method has also been performed for Escherichia coli (BW25113) and Staphylococcus aureus (ATCC, catalog number: 6538)]
LB broth Lennox (BD, catalog number: 240230 )
Ciprofloxacin hydrochloride (Corning Incorporated, catalog number: 61-277-RF )
Agar (BD, catalog number: 214040 )
MgCl₂^.7H₂O (VWR International, BDH^®, catalog number: 0288-VBD )
DI water (distilled water)
Sodium chloride (VWR International, BDH^®, catalog number: 0241-VBD )
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 )
Sterile Petri dishes
Microfuge tubes
Syringe 60 ml
Syringe needle (Gauge 21G)
Micropipettes (P1000, P200, P20)
Aluminum Foil
Saline (see Recipes)
Saline with Mg₂Cl₄^.7H₂O (see Recipes)
LB agar with Mg₂Cl₄^.7H₂O (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 )
General Purpose Laboratory Incubators, SHEL LAB (BioExpress, catalog number: G-1400-1 )
Nalgene^® Carboys with Handles, Polypropylene, Thermo Scientific (VWR International, catalog number: 16101-084 )
Nalgene^® Top Works^TM 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 )
Erlenmeyer Flasks (150 ml)
Homogenizer (PRO SCIENTIFIC, model: Bio-Gen Pro200 )
Table top refrigerated Centrifuge (Eppendorf, catalog number: 5418R )
Spectrophotometer (BD, catalog number: DU700 series )

Procedure

English

中文翻译

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如何引用

Marques, C. N. H. (2015). Isolation of Persister Cells from Biofilm and Planktonic Populations of Pseudomonas aeruginosa. Bio-protocol 5(18): e1590. DOI: 10.21769/BioProtoc.1590.