用于代谢物提取的假单胞菌和曲霉属真菌的生物膜共培养体系的建立

He Zheng; Nancy P. Keller; Yun Wang

doi:10.21769/BioProtoc.1667

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Peer-reviewed

Establishing a Biofilm Co-culture of Pseudomonas and Aspergillus for Metabolite Extraction

用于代谢物提取的假单胞菌和曲霉属真菌的生物膜共培养体系的建立

HZ He Zheng

NK Nancy P. Keller email

YW Yun Wang email

发布: 2015年12月05日第5卷第23期 DOI: 10.21769/BioProtoc.1667 浏览次数: 11491

评审: Arsalan DaudiTimo LehtiAnonymous reviewer(s)

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Jan 2015

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Abstract

Filamentous fungi and bacteria form mixed-species biofilms in nature and diverse clinical contexts (Frey-Klett et al., 2011; Peleg et al., 2010). The interactions between fungi and bacteria, often mediated by secreted metabolites, have important ramifications for the biology of the interacting partners (Frey-Klett et al., 2011). This is particularly true for the bacterium Pseudomonas aeruginosa (P. aeruginosa) and the fungus Aspergillus fumigatus (A. fumigatus) which often reside in the same niche such as lungs of cystic fibrosis (CF) patients. Some studies have reported that co-infection with P. aeruginosa and A. fumigatus could lead to a decrease in lung function relative to their respective single species infection (Amin et al., 2010; Peleg et al., 2010). Metabolite extraction and analysis allow for the characterization of specific microbial metabolites in the polymicrobial biofilm. This protocol describes how to prepare the Pseudomonas-Aspergillus co-culture biofilm on solid medium in preparation for metabolite extraction.

Keywords: Aspergillus fumigatus (烟曲霉)

Pseudomonas aeruginosa (铜绿假单胞菌)

Biofilm (生物膜)

Cross Kingdom communication (跨界交流)

Materials and Reagents

Plastic spreader (Excel Scientific, catalog number: 5PR-L-510 )
Falcon tubes (15 ml) (Corning Inc., catalog number: 352196 )
Petri dish (100 x 15 mm) (Corning Inc., catalog number: 351029 )
Petri dish (60 x 15 mm) (Corning Inc., catalog number: 351007 )
Inoculating needle (VWR International, catalog number: 12000-816 )
Glass vial (10 ml) (VWR International, catalog number: SC66022-300 )
Aluminum foil
Pseudomonas aeruginosa culture
Aspergillus fumigatus culture
D-glucose (Thermo Fisher Scientific, Alfa Aesar, catalog number: A1682836 )
Anhydrous dextrose (VWR International, BDH, catalog number: BDH9230500G )
Yeast extract (BD, Bacto^TM, catalog number: 288620 )
Agar-agar (Merck Millipore Corporation, catalog number: EM1.01614.0500 )
Peptone (BD, Bacto^TM, catalog number: 212120 )
Luria-Bertani (LB) dehydrated medium (Merck Millipore Corporation, catalog number: 1.10285.5000 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
GR ACS Sodium Nitrate (NaNO₃) (Merck Millipore Corporation, catalog number: SX06551 )
NaCl
Potassium Chloride (KCl) (VWR International, BDH, catalog number: 0395VBD500G )
Potassium dihydrogen phosphate (KH₂PO₄) (Thermo Fisher Scientific, Alfa Aesar, catalog number: 1159436 )
Boric acid (H₃BO₃) (Sigma-Aldrich, catalog number: B6768 )
Magnesium sulfate heptahydrate (MgSO₄^.7H₂O) (Sigma-Aldrich, catalog number: M1880 )
Copper(II) sulfate pentahydrate (CuSO₄^.5H₂O) (Sigma-Aldrich, catalog number: C8027 )
Zinc sulfate heptahydrate (ZnSO₄^.7H₂O) (Sigma-Aldrich, catalog number: Z0251 )
Ammonium molybdate tetrahydrate [(NH₄)₆Mo₇O₂₄^.4H₂O] (Sigma-Aldrich, catalog number: 431346 )
Manganese(II) sulfate monohydrate (MnSO₄^.H₂O) (Sigma-Aldrich, catalog number: M7634 )
Iron(II) sulfate heptahydrate (FeSO₄^.7H₂O) (Sigma-Aldrich, catalog number: F8633 )
Ethylenediaminetetraacetic acid disodium salt dehydrate (Na₂EDTA^.2H₂O) (Sigma-Aldrich, catalog number: E4884 )
Cobalt(II) chloride hexahydrate (CoCl₂^.6H₂O) (Sigma-Aldrich, catalog number: 255599 )
Tween 20 solution (Sigma-Aldrich, catalog number: P2287 )
Chloroform (VWR International, BDH, catalog number: PL049ZA4 )
Acetonitrile (Honeywell International Inc., catalog number: AH015-4 )
PBS-0.01 % Tween 20 buffer (see Recipes)
20x sodium nitrate salts solution (see Recipes)
Trace elements (see Recipes)
Medium recipes (see Recipes)
1. Solid Luria-Bertani (LB) dehydrated medium (see Recipes)
2. Solid yeast extract-peptone-dextrose medium (YPD) (see Recipes)

Equipment

Incubator (VWR International, model: 1535 )
Orbital shaker (Eppendorf AG, New Brunswick Scientific, model: I26 )
Centrifuge (Beckman, model: J20 ) with rotor (Beckman, model: JLA-10.500 )
Microscope (Celestron, model: 44345 )
Hemocytometer (Hausser Scientific, catalog number: 3500 )
Steel corer (38.1 mm in diameter) (Figure 1)

Figure 1. Steel corer
Steel blade (VWR International, catalog number: 55411-050 )
Steel pestle (VWR International, catalog number: 62400-336 )
0.2 μm-pore-size hydrophilic filter (VWR International, catalog number: 28145-477 )
Glass culture tube (15 ml) (VWR International, catalog number: 47729-583 )
Glass centrifuge tube (15 ml) (SP Industries, Wilmad-LabGlass, catalog number: C-8060-15 )
Nitrogen gas cylinder

Procedure

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如何引用

Zheng, H., Keller, N. P. and Wang, Y. (2015). Establishing a Biofilm Co-culture of Pseudomonas and Aspergillus for Metabolite Extraction. Bio-protocol 5(23): e1667. DOI: 10.21769/BioProtoc.1667.