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Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus

定量测定蓝藻细长聚球藻中作为生物膜形成代用指标的叶绿素

ES Eleonora Sendersky
RS Ryan Simkovsky
SG Susan S. Golden
RS Rakefet Schwarz

发布: 2017年07月20日第7卷第14期 DOI: 10.21769/BioProtoc.2406 浏览次数: 11405

评审: Dennis NürnbergMaria SinetovaAlexander Martin Ruecker

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Abstract

A self-suppression mechanism of biofilm development in the cyanobacterium Synechococcus elongatus PCC 7942 was recently reported. These studies required quantification of biofilms formed by mutants impaired in the biofilm-inhibitory process. Here we describe in detail the use of chlorophyll measurements as a proxy for biomass accumulation in sessile and planktonic cells of biofilm-forming strains. These measurements allow quantification of the total biomass as estimated by chlorophyll level and representation of the extent of biofilm formation by depicting the relative fraction of chlorophyll in planktonic cells.

Keywords: Biofilm (生物膜) search Cyanobacteria (蓝藻) search Synechococcus elongatus (细长聚球藻) search Chlorophyll measurement (叶绿素测定) search Sessile (无柄) search Planktonic (浮游) search

Background

Several recently published studies indicate an emerging interest in the mechanisms that underlie cell-aggregation and biofilm development in cyanobacteria (Fisher et al., 2013; Jittawuttipoka et al., 2013; Schatz et al., 2013; Enomoto et al., 2014; Schwarzkopf et al., 2014; Enomoto et al., 2015; Oliveira et al., 2015; Agostoni et al., 2016; Parnasa et al., 2016). We recently reported a self-biofilm-inhibitory mechanism that dictates planktonic growth of the model unicellular cyanobacterium Synechococcus elongatus PCC 7942 (Schatz et al., 2013; Nagar and Schwarz, 2015). Abrogation of the biofilm-inhibitory process by inactivation of particular genes results in robust biofilm development in this otherwise planktonic strain (Schatz et al., 2013; Nagar and Schwarz, 2015). These studies required quantification of the extent of biofilm development in various strains and under different conditions. Crystal violet is commonly used for quantification of biofilms in heterotrophic bacteria (O’Toole and Kolter, 1998). This staining procedure, however, quantifies only the sessile fraction of cells. Here we provide a detailed protocol for culture growth and quantification of cyanobacterial biofilms using chlorophyll measurement as a proxy for biomass accumulation in sessile as well as in planktonic cells. These measurements allow estimation of the total biomass accumulated and representation of the relative fraction of chlorophyll in sessile or in planktonic cells.

Materials and Reagents

  1. Custom-made Pyrex glass tubes for bacterial liquid cultures (200 x 32 mm, made from Pyrex tubing, Corning, catalog number: 8510-32-D)
    Note: Can order from http://www.degroot.co.il/.
  2. ‘Sponge plug’ (Plastic foam stoppers, 27 x 34 mm) (Jaece Industies, catalog number: L800-C )
  3. Pasteur pipettes (230 mm) (Romical, catalog number: 94-08401002 )
  4. 0.45 µm syringe filter (Sartorius, catalog number: 16555 )
  5. Filter Stericup-GP 250 ml Express Plus PES (0.22 μm) (EMD Millipore, catalog number: SCGPU05RE )
  6. Silicone tubing (6 x 9 mm, 4 x 6 mm) (Degania Silicone, catalog numbers: 2110600234 , 2110400434 , respectively)
  7. Sterile pipettes 1 and 25 ml
    1 ml pipettes (Corning, Costar®, catalog number: 4011 )
    25 ml pipettes (Corning, Costar®, catalog number: 4489)
  8. Sterilized pipette tips (Corning, Axygen®, catalog numbers: T-200-C , T-1000-C )
  9. Eppendorf tubes (1.5 ml) (Corning, Axygen®, catalog number: MCT-175-C )
  10. Synechococcus elongatus PCC7942
  11. Sodium nitrate (NaNO3) (Sigma-Aldrich, catalog number: S8170 )
  12. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Merck, catalog number: K26364082)
    Note: This product has been discontinued. Alternatively, Merck, catalog number: 105886 can be used.
  13. Calcium chloride dihydrate (CaCl2·2H2O) (ICN, catalog number: 10035-04-8)
    Note: This product has been discontinued. Alternatively, Bio-Lab, catalog number: 034205 can be used.
  14. Potassium phosphate dibasic (K2HPO4) (Honeywell International, Riedel-de-Haen, catalog number: 04248 )
  15. Ethylenediaminetetraacetic acid (Na2Mg·EDTA) (Bio-Lab, catalog number: 05142359)
    Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 051423 can be used.
  16. Ferric ammonium citrate (C6H11FeNO7) (MP Biomedicals, catalog number: 02158040 )
  17. Citric acid (C6H8O7) (Frutarom, catalog number: 2355511000)
    Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 030205 can be used.
  18. Boric acid (H3BO3) (Bio-Lab, catalog number: 02010591)
    Note: This product has been discontinued. Alternatively, Biosolve, catalog number: 020105 can be used.
  19. Manganese chloride hexahydrate (MnCl2·6H2O) (Duchefa Biochemie, catalog number: M0533 )
  20. Zinc sulfate heptahydrate (ZnSO4·7H2O) (CARLO ERBA Reagents, catalog number: 494907 )
  21. Sodium molybdate dihydrate (Na2MoO4·2H2O) (MP Biomedicals, catalog number: 194863 )
  22. Copper sulphate pentahydrate (CuSO4·5H2O) (Honeywell International, Riedel-de-Haen, catalog number: 12849 )
  23. Cobalt(II) nitrate hexahydrate (Co(NO3)2·6H2O) (Honeywell International, Riedel-de-Haen, catalog number: 12922)
    Note: This product has been discontinued. Alternatively, Sigma-Aldrich, catalog number: 239267 can be used.
  24. N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H3375 )
  25. Sodium hydroxide (NaOH) (Frutarom, catalog number: 5553510 )
  26. Acetone ((CH3)2CO), A.C.S. reagent (Avantor Performance Materials, J.T. Baker®, catalog number: 9006-03 )
  27. Sodium bicarbonate (NaHCO3) (DAEJUNG CHEMICAL & METALS, catalog number: 7566-4100 )
  28. Sodium thiosulfate (Na2S2O3) (Sigma-Aldrich, catalog number: 217247 )
  29. Bacto agar (BD, BactoTM, catalog number: 214010 )
  30. LB agar (Lennox) (BD, DifcoTM, catalog number: 240110 )
  31. Antibiotics (added as appropriate according to the resistance of the particular strain)
    1. Spectinomycin dihydrochloride pentahydrate (Duchefa Biochemie, catalog number: S0188 )
    2. Kanamycine sulphate monohydrate (Duchefa Biochemie, catalog number: K0126 )
    3. Gentamycin sulphate (Duchefa Biochemie, catalog number: G0124 )
    4. Chloramphenicol (Duchefa Biochemie, catalog number: C0113 )
  32. Liquid BG11-medium (see Recipes)
    1. Stock I (100x concentrated)
    2. Stock II (100x concentrated)
    3. Stock III (100x concentrated)
    4. Stock V (1,000x concentrated)
  33. Solid BG11-medium (see Recipes)

Equipment

  1. Laminar flow hood
  2. Fume hood
  3. Autoclave
  4. Spectrophotometer (Agilent Technologies, model: Cary 100 , catalog number: 10069000) and respective cuvettes (Cell type P.L = 10 mm/EA) (Starna Cells, catalog number: 9-SOG-10)
  5. Benchtop centrifuge (MiniSpin, max. centrifugal force: 12,100 x g) (Eppendorf, model: MiniSpin® , catalog number: 5452000018)
  6. Refrigerator (4-7 °C)
  7. Growth rooms (30 ± 2 °C and 24 ± 2 °C for liquid cultures and cultures on solid medium, respectively)
    Note: Growing cultures on solid medium at 24 °C rather than 30 °C allows maintaining the cultures for longer periods.
  8. Light source
    For liquid cultures–incandescent light producing a flux of 20-30 μmol photons m-2  sec-1; Cultures on solid-BG11 are illuminated by fluorescent light (5 μmol photons m-2  sec-1)
  9. Pipet-aid
  10. Single-channel pipettes 200 and 1,000 μl
  11. Bubbling system
    1. Air compressor (Oil free air compressor) (Assouline Compressors, model: vs 204 50 )
    2. Cylinder carbon dioxide (CO2, compr. 99.5%) (Maxima, catalog number: GCDCU2.527)
    3. Flow meters (Tuttnauer company, 0-0.1 L/min, 1-10 L/min)
    4. High-flow air pressure regulator (0-2 PSI) (Marsh Bellofram, model: Type 70 , catalog number: 960-129-000)
    Note: To bubble cultures with 3% CO2 in air the following setup is used (Figure 1): CO2 gas is mixed with compressed air using flow meters to yield 3% CO2 in air. This mixture is humidified by bubbling into 1.5 L double distilled water (DDW) in a 2 L bottle to prevent water evaporation during bubbling of the cultures. The outlet from this bottle is passed through an empty 2 L bottle (serving to trap residual liquid) and through an air pressure regulator (200 mbar is appropriate for bubbling of ~300 culture tubes). Bubbling of CO2 enriched air from the latter into multiple cultures is obtained using home-made manifolds constructed from silicone tubing and appropriate T-bar connectors. The gas in each manifold channel is passed through a 0.45 µm filter (Figure 1). A short video is provided to demonstrate the flow rate in individual cultures tubes (Video 1).


    Figure 1. A scheme describing the setup used to bubble cultures with CO2-enriched air

    Video 1. Cyanobacterial cultures under bubbling

Procedure

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© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Sendersky, E., Simkovsky, R., Golden, S. S. and Schwarz, R. (2017). Quantification of Chlorophyll as a Proxy for Biofilm Formation in the Cyanobacterium Synechococcus elongatus. Bio-protocol 7(14): e2406. DOI: 10.21769/BioProtoc.2406.

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微生物学 > 微生物生物膜 > 生物膜培养

细胞生物学 > 细胞分离和培养 > 细胞生长

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