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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   

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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

  1. Plastic spreader (Excel Scientific, catalog number: 5PR-L-510 )
  2. Falcon tubes (15 ml) (Corning Inc., catalog number: 352196 )
  3. Petri dish (100 x 15 mm) (Corning Inc., catalog number: 351029 )
  4. Petri dish (60 x 15 mm) (Corning Inc., catalog number: 351007 )
  5. Inoculating needle (VWR International, catalog number: 12000-816 )
  6. Glass vial (10 ml) (VWR International, catalog number: SC66022-300 )
  7. Aluminum foil
  8. Pseudomonas aeruginosa culture
  9. Aspergillus fumigatus culture
  10. D-glucose (Thermo Fisher Scientific, Alfa Aesar, catalog number: A1682836 )
  11. Anhydrous dextrose (VWR International, BDH, catalog number: BDH9230500G )
  12. Yeast extract (BD, BactoTM, catalog number: 288620 )
  13. Agar-agar (Merck Millipore Corporation, catalog number: EM1.01614.0500 )
  14. Peptone (BD, BactoTM, catalog number: 212120 )
  15. Luria-Bertani (LB) dehydrated medium (Merck Millipore Corporation, catalog number: 1.10285.5000 )
  16. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
  17. GR ACS Sodium Nitrate (NaNO3) (Merck Millipore Corporation, catalog number: SX06551 )
  18. NaCl
  19. Potassium Chloride (KCl) (VWR International, BDH, catalog number: 0395VBD500G )
  20. Potassium dihydrogen phosphate (KH2PO4) (Thermo Fisher Scientific, Alfa Aesar, catalog number: 1159436 )
  21. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768 )
  22. Magnesium sulfate heptahydrate (MgSO4.7H2O) (Sigma-Aldrich, catalog number: M1880 )
  23. Copper(II) sulfate pentahydrate (CuSO4.5H2O) (Sigma-Aldrich, catalog number: C8027 )
  24. Zinc sulfate heptahydrate (ZnSO4.7H2O) (Sigma-Aldrich, catalog number: Z0251 )
  25. Ammonium molybdate tetrahydrate [(NH4)6Mo7O24.4H2O] (Sigma-Aldrich, catalog number: 431346 )
  26. Manganese(II) sulfate monohydrate (MnSO4.H2O) (Sigma-Aldrich, catalog number: M7634 )
  27. Iron(II) sulfate heptahydrate (FeSO4.7H2O) (Sigma-Aldrich, catalog number: F8633 )
  28. Ethylenediaminetetraacetic acid disodium salt dehydrate (Na2EDTA.2H2O) (Sigma-Aldrich, catalog number: E4884 )
  29. Cobalt(II) chloride hexahydrate (CoCl2.6H2O) (Sigma-Aldrich, catalog number: 255599 )
  30. Tween 20 solution (Sigma-Aldrich, catalog number: P2287 )
  31. Chloroform (VWR International, BDH, catalog number: PL049ZA4 )
  32. Acetonitrile (Honeywell International Inc., catalog number: AH015-4 )
  33. PBS-0.01 % Tween 20 buffer (see Recipes)
  34. 20x sodium nitrate salts solution (see Recipes)
  35. Trace elements (see Recipes)
  36. Medium recipes (see Recipes)
    1. Solid Luria-Bertani (LB) dehydrated medium (see Recipes)
    2. Solid yeast extract-peptone-dextrose medium (YPD) (see Recipes)

Equipment

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


    Figure 1. Steel corer

  7. Steel blade (VWR International, catalog number: 55411-050 )
  8. Steel pestle (VWR International, catalog number: 62400-336 )
  9. 0.2 μm-pore-size hydrophilic filter (VWR International, catalog number: 28145-477 )
  10. Glass culture tube (15 ml) (VWR International, catalog number: 47729-583 )
  11. Glass centrifuge tube (15 ml) (SP Industries, Wilmad-LabGlass, catalog number: C-8060-15 )
  12. Nitrogen gas cylinder

Procedure

  1. Biofilm co-culture of Pseudomonas and Aspergillus
    1. Pseudomonas aeruginosa culture preparation
      1. Streak-inoculate Pseudomonas aeruginosa PA14 strain from -80 °C glycerol stock onto solid LB medium in a 100 x 15 mm Petri dish and incubate at 37 °C for 24 h.
      2. Immediately prior to use for co-culture experiments, inoculate 5 ml of liquid LB medium in a 15 ml glass culture tube with a single colony from PA14 LB solid medium culture in step A1a and incubate at 37 °C, 225 rpm for 14 h to early stationary phase.
    2. Aspergillus fumigatus conidia preparation
      1. Take a ~10 μl plug from the -80 °C glycerol stock of Aspergillus fumigatus AF293 using a sterile inoculating needle and streak onto solid glucose minimal medium (GMM) in a 100 x 15 mm Petri dish and incubate at 37 °C for 5 days (Figure 2).


        Figure 2. Example of A. fumigatus AF293 streak culture after 5 days of incubation at 37 °C

      2. Immediately prior to use, add 5 ml of phosphate buffered saline (PBS) -0.01% Tween 20 buffer (pH 7.2) to the fungal plate and gently scrap the surface of fungal colony with a sterile, plastic spreader.
      3. Collect the conidia suspension from Petri dish and transfer into a 15 ml Falcon tube.
      4. Centrifuge the conidia suspension at 5,000 x g for 5 min. Discard supernatant and re-suspend conidia pellet with 5 ml of PBS-0.01% Tween 20 buffer.
      5. Dilute the conidia suspension with PBS-0.01% Tween 20 buffer to a final concentration between 1 to 2 x 106 conidia/ml. Quantify the number of conidia using a hemocytometer (Figure 3).


        Figure 3. Example of A. fumigatus conidia under microscope

    3. Pseudomonas and Aspergillus co-culture biofilm preparation
      1. Pour 10 ml of autoclaved YPD solid medium (1.6% agar) (bottom layer) into a 60 x 15 mm Petri dish and solidify at room temperature.
        Note: Prepare multiple YPD agar plates for different co-culture time points and replicates.
      2. Autoclave YPD solid medium (0.5% agar) (top layer) and cool in 50 °C water bath for 15 min.
      3. After YPD top layer is cooled, inoculate with freshly prepared and vortex-mixed Aspergillus conidia suspension to a final concentration of 3.33 x 105 conidia/ml.
      4. Mix the YPD top layer containing conidia thoroughly and pour 3 ml of the mixed top layer onto the solidified bottom layer.
        Note: High temperature of autoclaved agar will kill Aspergillus conidia. Thus, it is critical to ensure that the temperature of YPD top layer is cooled enough to the extent that medium bottle can be held with hands. In the meanwhile, agar will solidify if the temperature drops too much. Steps A3c-d should be done as quickly as possible.
      5. Once the top layer is solidified, incubate the plate (fungal biofilm facing up) at 25 °C for 12 h.
        Note: A. fumigatus grows slower than P. aeruginosa. 12 h pre-culture is necessary to avoid overwhelming bacterial growth.
      6. Spot-inoculate 10 μl aliquots of freshly prepared early stationary phase P. aeruginosa PA14 LB liquid culture in the center of the performed fungal lawn.
      7. Incubate the co-culture biofilm (co-culture biofilm facing up) at 25 °C for an additional 7 days.
        Notes:
        1. See Figure 5 for an example of the co-culture biofilm.
        2. It is critical to synchronize the growth of P. aeruginosa (24 h) and A. fumigatus (5 d) prior to the preparation of co-culture biofilm.

  2. Metabolite extraction
    1. After different periods of growth (for example, every two days after inoculation), cut the co-culture biofilm with a sterile steel corer (38.1 mm in diameter), which encompasses the co-culture biofilm.
    2. Cut the 38.1 mm diameter extraction core into four, equal quarters using a sterile blade.
    3. Transfer one quarter (about 285 mm2 of surface) to a glass culture tube.
      Note: Transfer both co-culture biofilm and supporting agar (Figure 4).


      Figure 4. Example of cutting co-culture plate for extraction

    4. Add 4 ml milli-Q water and homogenize using a pestle.
    5. Add 4 ml chloroform, cap the tube, and vortex vigorously for 15 min.
    6. Transfer the mixture to a 15 ml glass centrifuge tube.
    7. Centrifuge at 500 x g for 10 min.
    8. Collect water phase supernatant and filter through a 0.2 μm-pore-size hydrophilic filter.
    9. Collect 1.5 ml of chloroform phase to a 10 ml glass vial.
    10. Evaporate chloroform to dryness by using a gentle flow of nitrogen.
    11. Re-dissolve the dried extract with 1.5 ml mixture of 50/50% acetonitrile/water solution.
      Note: This dissolution solvent can be used for the assessment of metabolites that have similar chemical property as Pseudomonas phenazines, which were analyzed in Zheng et al. (2015).
    12. If extracted sample contains metabolites that are unstable in acetonitrile such as 5-methoxy-phenazine-1-carboxylic acid (5-Me-PCA), collect another 1.5 ml aliquot of chloroform phase after step B9, evaporate to dryness under a gentle flow of nitrogen and re-dissolve in 1.5 ml milli-Q water.
      Note: Most bacterial and fungal metabolites are light sensitive, thus, it is strongly suggested to protect extracts from light by wrapping all containers with aluminum foil.

Representative data


Figure 5. Representative image of the co-culture biofilm of Pseudomonas aeruginosa PA14 wild-type (center colony) and Aspergillus fumigatus AF293 wild-type (wrinkled biofilm surrounding bacterial colony) after 6 days of incubation at 25 °C

Notes

  1. It is critical to keep the same pre-culture condition for both bacteria and fungi before co-culture inoculation. For fungi, you need to ensure the same pre-culture time. For bacteria, early stationary phase culture is recommended in this experiment. OD600 can be measured to check bacteria growth. If you use different fungal and/or bacterial strains, it is strongly recommended to do a series of co-cultures with different pre-culture time to determine a condition that gives the best result.
  2. Co-culture biofilms can grow in various Petri dishes of different sizes. You can scale up or down the dimension of co-cultures based on your research need. Note that the co-culture biofilm growth may vary in different types of Petri dishes.

Recipes

  1. PBS-0.01% Tween 20 buffer (1 L)
    NaCl
    8 g
    KCl
    0.2 g
    Na2HPO4
    1.42 g
    KH2PO4
    0.24 g
    Tween 20 solution
    0.1 ml
    Mix in about 900 ml milli-Q water and adjust pH to 7.2 with 5 M NaOH solution
    Adjust volume with milli-Q water to 1 L and autoclave
  2. 20x sodium nitrate salts solution
    NaNO3
    120 g
    KCl
    10.4 g
    MgSO4.7H2O
    10.4 g
    KH2PO4
    30.4 g
    Add milli-Q water up to 1 L
    Autoclave and store at room temperature
  3. Trace elements (100 ml)
    ZnSO4.7H2O
    2.2 g
    H3BO3
    1.1 g
    MnSO4.H2O
    0.43 g
    FeSO4.7H2O
    0.5 g
    CoCl2.6H2O
    0.17 g
    CuSO4.5H2O
    0.16 g
    (NH4)6Mo7O24.4H2O
    0.11g
    Na4EDTA.2H2O
    4.9 g
    Add solids into 80 ml milli-Q water, dissolving each completely before adding the next
    Add 20 ml of 1 M NaOH solution to adjust the pH to 6.5
    Mix solution until solids are completely dissolved
  4. Medium recipes
    1. Solid glucose minimal medium (GMM) (1 L)
      20x sodium nitrate salts
      50 ml
      Trace elements (mix before using)
      1 ml
      D-glucose
      10 g
      Agar-agar
      16 g
      Mix in 900 ml milli-Q water and adjust pH to 6.5 with 5 M NaOH solution
      Adjust volume with Milli-Q water to 1 L
    2. Solid yeast extract-peptone-dextrose medium (YPD) (1 L)
      Anhydrate dextrose
      20 g
      Yeast extract
      10 g
      Bacto peptone
      20 g
      Agar-agar
      16 g
      Trace elements (mix before using)
      1 ml
      Mix in 900 ml milli-Q water, adjust volume with milli-Q water to 1 L, and autoclave
    3. Solid Luria-Bertani (LB) dehydrated medium (1 L)
      Luria-Bertani medium powder
      25 g
      *Agar-agar
      16 g
      Mix in 900 ml milli-Q water, adjust volume with milli-Q water to 1 L, and autoclave
      Note: *Do not add agar when preparing liquid LB medium.

Acknowledgments

This work was supported by startup and ISEN funding from Northwestern University (to Y. W.), NIH Grant R01 GM 067725 (to N. L. K.), and National Science Foundation Grant Emerging Frontiers in Research and Innovation 1136903 (to N. P. K.). This protocol was adapted from Zheng et al. (2015).

References

  1. Amin, R., Dupuis, A., Aaron, S. D. and Ratjen, F. (2010). The effect of chronic infection with Aspergillus fumigatus on lung function and hospitalization in patients with cystic fibrosis. Chest 137(1): 171-176.
  2. Frey-Klett, P., Burlinson, P., Deveau, A., Barret, M., Tarkka, M. and Sarniguet, A. (2011). Bacterial-fungal interactions: hyphens between agricultural, clinical, environmental, and food microbiologists. Microbiol Mol Biol Rev 75(4): 583-609.
  3. Peleg, A. Y., Hogan, D. A. and Mylonakis, E. (2010). Medically important bacterial-fungal interactions. Nat Rev Microbiol 8(5): 340-349.
  4. Shimizu, K. and Keller, N. P. (2001). Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics 157(2): 591-600.
  5. Zheng, H., Kim, J., Liew, M., Yan, J. K., Herrera, O., Bok, J. W., Kelleher, N. L., Keller, N. P. and Wang, Y. (2015). Redox metabolites signal polymicrobial biofilm development via the NapA oxidative stress cascade in Aspergillus. Curr Biol 25(1): 29-37.

简介

丝状真菌和细菌在自然界和不同的临床背景中形成混合物种生物膜(Frey-Klett等人,2011; Peleg等人,2010)。真菌和细菌之间的相互作用,通常由分泌的代谢物介导,对相互作用的伴侣的生物学具有重要的影响(Frey-Klett等人,2011)。对于绿脓假单胞菌(铜绿假单胞菌)和真菌烟曲霉( A。fumigatus >),其常常位于相同的位置,例如囊性纤维化(CF)患者的肺。一些研究报道了与p的共感染。铜绿和 烟曲霉可能导致相对于它们各自的单一物种感染的肺功能降低(Amin等人,2010; Peleg等人,2010)。代谢物提取和分析允许表征多微生物生物膜中的特定微生物代谢物。该方案描述了如何在固体培养基上制备假单胞菌属 - 曲霉菌共培养生物膜以准备代谢物提取。

关键字:烟曲霉, 铜绿假单胞菌, 生物膜, 跨界交流

材料和试剂

  1. 塑料撒布机(Excel Scientific,目录号:5PR-L-510)
  2. Falcon管(15ml)(Corning Inc.,目录号:352196)
  3. 培养皿(100×15mm)(Corning Inc.,目录号:351029)
  4. 培养皿(60×15mm)(Corning Inc.,目录号:351007)
  5. 接种针(VWR International,目录号:12000-816)
  6. 玻璃小瓶(10ml)(VWR International,目录号:SC66022-300)
  7. 铝箔
  8. 绿脓假单胞菌培养物
  9. 烟曲霉文化
  10. D-葡萄糖(Thermo Fisher Scientific,Alfa Aesar,目录号:A1682836)
  11. 无水葡萄糖(VWR International,BDH,目录号:BDH9230500G)
  12. 酵母提取物(BD,Bacto TM ,目录号:288620)
  13. 琼脂(Merck Millipore Corporation,目录号:EM1.01614.0500)
  14. 蛋白胨(BD,Bacto TM ,目录号:212120)
  15. Luria-Bertani(LB)脱水培养基(Merck Millipore Corporation,目录号:1.10285.5000)
  16. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:S5881)
  17. GR ACS硝酸钠(NaNO 3)(Merck Millipore Corporation,目录号:SX06551)
  18. NaCl
  19. 氯化钾(KCl)(VWR International,BDH,目录号:0395VBD500G)
  20. 磷酸二氢钾(KH 2 PO 4)(Thermo Fisher Scientific,Alfa Aesar,目录号:1159436)
  21. 硼酸(H 3 BO 3)(Sigma-Aldrich,目录号:B6768)
  22. 硫酸镁七水合物(MgSO 4)7H 2 O(Sigma-Aldrich,目录号:M1880)
  23. 硫酸铜(II)五水合物(CuSO 4·5H 2 O·5H 2 O)(Sigma-Aldrich,目录号:C8027)
  24. 硫酸锌七水合物(ZnSO 4·7H 2 O 7H 2 O)(Sigma-Aldrich,目录号:Z0251)
  25. 钼酸铵四水合物[(NH 4)6 Mo 6 Mo 7 O 24] 4H 2 O](Sigma-Aldrich,目录号:431346)
  26. 硫酸锰(II)一水合物(MnSO 4·H 2·H 2 O)(Sigma-Aldrich,目录号:M7634)
  27. 硫酸铁(II)七水合物(FeSO 4·7H 2 O 7H 2 O)(Sigma-Aldrich,目录号:F8633)
  28. 乙二胺四乙酸二钠盐二水合物(Na 2 EDTA二钠2H 2 O)(Sigma-Aldrich,目录号:E4884)
  29. 氯化钴(II)六水合物(CoCl 2·6H 2 O,6H 2 O)(Sigma-Aldrich,目录号:255599)
  30. 吐温20溶液(Sigma-Aldrich,目录号:P2287)
  31. 氯仿(VWR International,BDH,目录号:PL049ZA4)
  32. 乙腈(Honeywell International Inc.,目录号:AH015-4)
  33. PBS-0.01%吐温20缓冲液(参见配方)
  34. 20x硝酸钠溶液(见配方)
  35. 微量元素(参见配方)
  36. 中等配方(参见配方)
    1. 固体Luria-Bertani(LB)脱水培养基(参见配方)
    2. 固体酵母提取物 - 蛋??白胨 - 葡萄糖培养基(YPD)(参见Recipes)

设备

  1. 孵化器(VWR International,型号:1535)
  2. 轨道摇床(Eppendorf AG,New Brunswick Scientific,型号:I26)
  3. 用转子(Beckman,型号:JLA-10.500)离心(Beckman,型号:J20)
  4. 显微镜(Celestron,型号:44345)
  5. 血细胞计数器(Hausser Scientific,目录号:3500)
  6. 钢芯(直径38.1mm)(图1)


    图1. Steel corer

  7. 钢刀片(VWR International,目录号:55411-050)
  8. 钢杵(VWR International,目录号:62400-336)
  9. 0.2μm孔径亲水性过滤器(VWR International,目录号:28145-477)
  10. 玻璃培养管(15ml)(VWR International,目录号:47729-583)
  11. 玻璃离心管(15ml)(SP Industries,Wilmad-LabGlass,目录号:C-8060-15)
  12. 氮气瓶

程序

  1. 假单胞菌和曲霉菌的生物膜共培养
    1. 绿脓杆菌培养准备
      1. 将来自-80℃甘油原液的条纹接种绿脓杆菌 PA14菌株在固体上 ?LB培养基在100×15mm培养皿中,并在37℃孵育24小时
      2. 在即将用于共培养实验之前,接种5ml 的液体LB培养基在具有单个菌落的15ml玻璃培养管中 来自步骤A1a中的PA14LB固体培养基,并在37℃,225℃下孵育 ?rpm 14小时至早期稳定期。
    2. 烟曲霉分生孢子制剂
      1. 从?10℃的曲霉菌的甘油储备液中取出约10μl的塞子 烟曲霉AF293,使用无菌接种针并在固体上划线 ?葡萄糖基本培养基(GMM)在100×15mm培养皿中并孵育 在37℃下培养5天(图2)

        图2. A的示例。烟曲霉 AF293条纹培养在37℃孵育5天后

      2. 使用前,立即加入5ml磷酸盐缓冲盐水 (PBS)-0.01%吐温20缓冲液(pH7.2)加入到真菌平板中并温和 用无菌塑料撒布机刮除真菌菌落的表面。
      3. 从培养皿收集分生孢子悬浮液,并转移到15毫升Falcon管。
      4. 在5,000×g离心分生孢子悬浮液5分钟。丢弃 上清液并用5ml PBS-0.01%Tween再悬浮分生孢子沉淀 20缓冲液。
      5. 用PBS-0.01%吐温20稀释分生孢子悬浮液 ?缓冲液至终浓度为1至2×10 6分生孢子/ml。 使用血细胞计数器量化分生孢子数(图3)。


        图3. A的示例。烟曲霉显微镜下的分生孢子

    3. 假单胞菌和曲霉菌共培养生物膜制剂
      1. 倒入10ml高压灭菌的YPD固体培养基(1.6%琼脂)(底层) ?装入60×15mm培养皿中并在室温下固化。
        注意:准备多个YPD琼脂平板用于不同的共培养时间点和重复。
      2. 高压灭菌YPD固体培养基(0.5%琼脂)(顶层),在50℃水浴中冷却15分钟。
      3. 在YPD顶层冷却后,接种新鲜制备的和 涡旋混合的曲霉属分生孢子悬浮液至终浓度 3.33×10 5分生孢子/ml
      4. 混合含有分生孢子的YPD顶层,并将3ml混合的顶层倒在固化的底层上。
        注意:高温灭菌琼脂的高温会杀死曲霉菌 分生孢子。因此,关键是确保YPD的温度 顶层被冷却到中等瓶可以的程度 举行用手。同时,如果温度,琼脂将凝固 ?滴太多。步骤A3c-d应尽快完成。
      5. 一旦顶层固化,孵化板(真菌生物膜面向上)在25℃下12小时。
        注意:烟曲霉生长慢于铜绿假单胞菌。 12 h预培养是必要的,以避免压倒性的细菌生长。
      6. 点接种新鲜制备的10微升等分固定 相位P。铜绿色 PA14 LB液体培养在中心 进行真菌草坪
      7. 将共培养生物膜(共培养生物膜面朝上)在25°C孵育另外7天。
        注意:
        1. 见图5的共培养生物膜的例子。
        2. 关键是同步铜绿假单胞菌的生长(24小时) 和烟曲霉(5),在制备共培养生物膜之前

  2. 代谢物提取
    1. 经过不同的生长期(例如,每两天后 接种),用无菌钢芯钳切割共培养生物膜 (直径38.1mm),其包括共培养生物膜
    2. 使用无菌刀片将38.1 mm直径的提取芯切割成四等分四分之一
    3. 将四分之一(约285mm 2的表面)转移到玻璃培养管 注意:转移共培养生物膜和支持琼脂(图4)。


      图4.切割共培养板的示例

    4. 加入4ml milli-Q水,并用研杵匀浆
    5. 加入4ml氯仿,盖上试管,并剧烈涡旋15分钟
    6. 将混合物转移到15ml玻璃离心管中
    7. 以500×g离心10分钟。
    8. 收集水相上清液,并通过0.2μm孔径的亲水性过滤器过滤
    9. 收集1.5ml氯仿相到10ml玻璃小瓶中
    10. 使用温和的氮气流将氯仿蒸发至干
    11. 用1.5ml 50/50%乙腈/水溶液的混合物重新溶解干燥的提取物。
      注意:此溶解溶剂可用于评估 具有与假单胞菌相似化学性质的代谢物 吩嗪,其在Zheng等人(2015)。
    12. 如果 提取的样品含有在乙腈中不稳定的代谢物 例如5-甲氧基 - 吩嗪-1-羧酸(5-Me-PCA) 在步骤B9之后另一个1.5ml等分的氯仿相,蒸发至 在温和的氮气流下干燥并重新溶解于1.5ml milli-Q水。
      注意:大多数细菌和真菌代谢物是轻的 ?敏感,因此,强烈建议保护提取物 通过用铝箔包装所有容器来照亮。

代表数据


图5.铜绿假单胞菌PA14野生型(中心菌落)和烟曲霉AF393野生型(皱纹生物膜周围环境)的共培养生物膜的代表性图像细菌菌落)在25℃孵育6天后

笔记

  1. 在共培养接种之前保持细菌和真菌的相同预培养条件是至关重要的。对于真菌,你需要确保相同的预培养时间。对于细菌,在本实验中推荐早期的固定相培养。 OD 600可以测量以检查细菌生长。如果使用不同的真菌和/或细菌菌株,强烈建议在不同的预培养时间进行一系列的共培养,以确定获得最佳结果的条件。
  2. 共培养生物膜可以在不同尺寸的各种培养皿中生长。您可以根据您的研究需要,扩大或缩小共同文化的维度。注意,共培养生物膜生长可以在不同类型的培养皿中变化

食谱

  1. PBS-0.01%吐温20缓冲液(1L)
    NaCl
    8克
    KCl
    0.2 g
    Na HPO 4
    1.42克
    KH 2 PO 4
    0.24克
    吐温20溶液
    0.1 ml
    在约900ml milli-Q水中混合并用5M NaOH溶液将pH调节至7.2 用milli-Q水调节体积至1升,高压釜
  2. 20x硝酸钠溶液
    NaNO 3
    120克
    KCl
    10.4克
    MgSO 4。 。 O
    10.4克
    KH 2 PO 4
    30.4克
    添加milli-Q水至1 L
    高压灭菌并在室温下贮存
  3. 微量元素(100 ml)
    ZnSO 4 。 7H O
    2.2 g
    H 3 BO 3
    1.1克
    MnSO 4 H sub 2 O
    0.43克
    FeSO 4 7H <2> O
    0.5克
    CoCl <2> 6H <2> O
    0.17克
    CuSO 4 5H sub 2 O
    0.16 g
    (NH 4)6 Mo 7+ O 24+。在本发明的一个实施方案中, O
    0.11g
    Na EDTA 2H 2 4.9克
    将固体加入到80ml milli-Q水中,在添加下一个
    之前完全溶解 加入20ml 1M NaOH溶液,将pH调节至6.5 混合溶液直到固体完全溶解
  4. 中等配方
    1. 固体葡萄糖基本培养基(GMM)(1L)
      20x硝酸钠盐 50 ml
      微量元素(使用前混合)
      1 ml
      D-葡萄糖
      10克
      琼脂
      16克
      在900ml milli-Q水中混合并用5M NaOH溶液调节pH至6.5 用Milli-Q水调节音量至1 L
    2. 固体酵母提取物 - 蛋??白胨 - 葡萄糖培养基(YPD)(1L)
      无水葡萄糖
      20克
      酵母提取物
      10克
      细菌蛋白胨
      20克
      琼脂
      16克
      微量元素(使用前混合)
      1 ml
      在900ml milli-Q水中混合,用milli-Q水调节体积至1L,并用高压釜
    3. 固体Luria-Bertani(LB)脱水培养基(1L)
      Luria-Bertani中等粉末
      25克
      *琼脂
      16克
      在900ml milli-Q水中混合,用milli-Q水调节体积至1L,并用高压釜
      注意:*在制备液体LB培养基时不要添加琼脂。

致谢

这项工作是由来自西北大学(至Y. W.),NIH Grant R01 GM 067725(至N. L. K.)和国家科学基金会新兴前沿研究和创新1136903(到N. P. K.)的启动和ISEN资助支持的。方案改编自Zheng等人(2015)。

参考文献

  1. Amin,R.,Dupuis,A.,Aaron,S.D.and Ratjen,F。(2010)。 使用 Aspergillus fumigatus的慢性感染对肺功能和住院的影响囊性纤维化患者 胸部 137(1):171-176。
  2. Frey-Klett,P.,Burlinson,P.,Deveau,A.,Barret,M.,Tarkka,M。和Sarniguet,A。(2011)。 细菌 - 真菌相互作用:农业,临床,环境和食品微生物学家之间的连字符。 Microbiol Mol Biol Rev 75(4):583-609。
  3. Peleg,A.Y.,Hogan,D.A。和Mylonakis,E。(2010)。 医学上重要的细菌 - 真菌相互作用 Nat Rev Microbiol 8(5):340-349。
  4. Shimizu,K。和Keller,N.P。(2001)。 cAMP依赖性蛋白激酶在调节形态和化学过渡的G蛋白信号转导途径中的遗传参与 Aspergillus nidulans 。 Genetics 157(2):591-600。
  5. 正在加载...关于我们|联系我们|免责声明|网站地图|友情链接Copyright?2016焦点科技。版权所有氧化还原代谢物通过曲霉菌中的NapA氧化应激级联反应信号多微生物生物膜发育 。 Curr Biol 25(1):29-37。
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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.
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