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Advanced Techniques for In Situ Analysis of the Biofilm Matrix (Structure, Composition, Dynamics) by Means of Laser Scanning Microscopy

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Microbial Biofilms

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1147))

Abstract

The extracellular constituents in bioaggregates and biofilms can be imaged four dimensionally by using laser scanning microscopy. In this protocol we provide guidance on how to examine the various extracellular compartments in between microbial cells and communities associated with interfaces. The current options for fluorescence staining of matrix compounds and extracellular microhabitats are presented. Furthermore, practical aspects are discussed and useful notes are added. The chapter ends with a brief introduction to other approaches for EPS analysis and an outlook for future needs.

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References

  1. Wingender J, Neu TR, Flemming H-C (1999) Microbial extracellular polymeric substances. Springer, Heidelberg

    Google Scholar 

  2. Neu TR, Lawrence JR (2009) Extracellular polymeric substances in microbial biofilms. In: Moran A, Brenan P, Holst O, von Itzstein M (eds) Microbial glycobiology: structures, relevance and applications. Elsevier, San Diego, pp 735–758

    Google Scholar 

  3. Flemming HC, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8:623–633

    PubMed  CAS  Google Scholar 

  4. Allison DG, Sutherland IW, Neu TR (2003) EPS: what’s in an acronym? In: Mc Bain A, Allison DG, Brading M, Rickard A, Verran J, Walker J (eds) Biofilm communities: order from chaos? BioLine, Cardiff, pp 381–387

    Google Scholar 

  5. Miltner A, Bombach P, Schmidt-Brücken B, Kästner M (2012) SOM genesis: microbial biomass as a significant source. Biogeochemistry 111:41–55

    CAS  Google Scholar 

  6. Schermelleh L, Heintzmann R, Leonhardt H (2010) A guide to super-resolution fluorescence microscopy. J Cell Biol 190:165–175

    PubMed Central  PubMed  CAS  Google Scholar 

  7. Lawrence JR, Swerhone GDW, Leppard GG et al (2003) Scanning transmission x-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms. Appl Environ Microbiol 69:5543–5554

    PubMed Central  PubMed  CAS  Google Scholar 

  8. Liu X, Eusterhues K, Thieme J et al (2013) STXM and NanoSIMS investigations on EPS fractions before and after adsorption to goethite. Environ Sci Technol 47:3158–3166

    PubMed  CAS  Google Scholar 

  9. Behrens S, Kappler A, Obst M (2012) Linking environmental processes to the in situ functioning of microorganisms by high-resolution secondary ion mass spectrometry (NanoSIMS) and scanning transmission X-ray microscopy (STXM). Environ Microbiol 14:2851–2869

    PubMed  CAS  Google Scholar 

  10. Remusat L, Hatton PJ, Nico PS et al (2012) NanoSIMS study of organic matter associated with soil aggregates: advantages, limitations, and combination with STXM. Environ Sci Technol 46:3943–3949

    PubMed  CAS  Google Scholar 

  11. Rasconi S, Jobard M, Jouve L, Sime-Ngando T (2009) Use of calcofluor white for detection, identification, and quantification of phytoplanktonic fungal parasites. Appl Environ Microbiol 75:2545–2553

    PubMed Central  PubMed  CAS  Google Scholar 

  12. Neu TR, Marshall KC (1991) Microbial “footprints”: a new approach to adhesive polymers. Biofouling 3:101–112

    Google Scholar 

  13. Neu TR (1992) Microbial “footprints” and the general ability of microorganisms to label interfaces. Can J Microbiol 38:1005–1008

    Google Scholar 

  14. Sharon N, Lis H (2003) Lectins. Kluwer Academic Publishers, Dordrecht

    Google Scholar 

  15. Schloter M, Borlinghaus R, Bode W, Hartmann A (1993) Direct identification, and localization of Azospirillum in the rhizosphere of wheat using fluorescence-labelled monoclonal antibodies and confocal scanning laser microscopy. J Microsc 171:173–177

    Google Scholar 

  16. Assmus B, Schloter M, Kirchhof G et al (1997) Improved in situ tracking of rhizosphere bacteria using dual staining with fluorescence-labeled antibodies and rRNA-targeted oligonucleotides. Microb Ecol 33:32–40

    PubMed  Google Scholar 

  17. Schloter M, Wiehe W, Assmus B et al (1997) Root colonization of different plants by plant-growth-promoting Rhizobium leguminosarum bv. trifolii R39 studied with monospecific polyclonal antisera. Appl Environ Microbiol 63:2038–2046

    PubMed Central  PubMed  CAS  Google Scholar 

  18. Gilbert B, Assmus B, Hartmann A, Frenzel P (1998) In situ localization of two methanotrophic strains in the rhizosphere of rice plants. FEMS Microbiol Ecol 25:117–128

    CAS  Google Scholar 

  19. Palmer RJ Jr, Gordon SM, Cisar JO, Kolenbrander PE (2003) Coaggregation-mediated interactions of Streptococci and Actinomyces detected in initial human dental plague. J Bacteriol 185:3400–3409

    PubMed  CAS  Google Scholar 

  20. Chalmers NI, Palmer RJ Jr, Du-Thumm L et al (2007) Use of quantum dot luminescent probes to achieve single: cell resolution of human oral bacteria in biofilms. Appl Environ Microbiol 73:630–636

    PubMed Central  PubMed  CAS  Google Scholar 

  21. Periasamy S, Chalmers NI, Du-Thumm L, Kolenbrander PE (2009) Fusobacterium nucleatum ATCC 10953 requires Actinomyces naeslundii ATCC 43146 for growth on saliva in a three-species community that includes Streptococcus oralis 34. Appl Environ Microbiol 75:3250–3257

    PubMed Central  PubMed  CAS  Google Scholar 

  22. Periasamy S, Kolenbrander PE (2009) Mutualistic biofilm communities develop with Porphyromonas gingivalis and initial, early and late colonizers of enamel. J Bacteriol 191:6804–6811

    PubMed Central  PubMed  CAS  Google Scholar 

  23. Periasamy S, Kolenbrander PE (2010) Central role of the early colonizer Veillonella sp. in establishing multispecies biofilm communities with initial, middle, and late colonizers of enamel. J Bacteriol 192:2965–2972

    PubMed Central  PubMed  CAS  Google Scholar 

  24. Neu TR, Lawrence JR (1999) Lectin-binding-analysis in biofilm systems. Methods Enzymol 310:145–152

    PubMed  CAS  Google Scholar 

  25. Neu TR, Swerhone GDW, Lawrence JR (2001) Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems. Microbiology 147:299–313

    PubMed  CAS  Google Scholar 

  26. Staudt C, Horn H, Hempel DC, Neu TR (2003) Screening of lectins for staining lectin-specific glycoconjugates in the EPS of biofilms. In: Lens P, Moran AP, Mahony T, Stoodley P, O’Flaherty V (eds) Biofilms in medicine, industry and environmental technology. IWA Publishing, UK, pp 308–327

    Google Scholar 

  27. Zippel B, Neu TR (2011) Characterization of glycoconjugates of extracellular polymeric substances in tufa-associated biofilms by using fluorescence lectin-binding analysis. Appl Environ Microbiol 77:505–516

    PubMed Central  PubMed  CAS  Google Scholar 

  28. Lu S, Chourey K, Reiche M et al (2013) Insights into the structure and metabolic function of microbes that shape pelagic iron-rich aggregates (“iron snow”). Appl Environ Microbiol 79:4272–4281

    PubMed Central  PubMed  CAS  Google Scholar 

  29. Bennke CM, Neu TR, Fuchs BM, Amann R (2013) Mapping glycoconjugate-mediated interactions of marine Bacteroidetes with diatoms. Syst Appl Microbiol 36:417–425

    PubMed  CAS  Google Scholar 

  30. Lawrence JR, Swerhone GDW, Kuhlicke U, Neu TR (2007) In situ evidence for microdomains in the polymer matrix of bacterial microcolonies. Can J Microbiol 53:450–458

    PubMed  CAS  Google Scholar 

  31. Saarima C, Peltola M, Raulio M et al (2006) Characterisation of adhesion threads of Deinococcus geothermalis as Type IV pili. J Bacteriol 188:7016–7021

    Google Scholar 

  32. Peltola M, Neu TR, Kanto-Oqvist L et al (2008) Architecture of Deinococcus geothermalis biofilms on glass and steel: a lectin study. Environ Microbiol 10:1752–1759

    PubMed  CAS  Google Scholar 

  33. Zubkov MV, Fuchs BM, Eilers H et al (1999) Determination of total protein content of bacterial cells by SYPRO staining and flow cytometry. Appl Environ Microbiol 65:3251–3257

    PubMed Central  PubMed  CAS  Google Scholar 

  34. Hinck S, Mußmann M, Salman V et al (2011) Vacuolated Beggiatoa-like filaments from different hypersaline environments form a novel genus. Environ Microbiol 13:3194–3205

    PubMed  Google Scholar 

  35. Rusznyak A, Akob DM, Nietzsche S et al (2012) Calcite biomineralization by bacterial isolates from the recently discovered pristine karstic Herrenberg cave. Appl Environ Microbiol 78:1157–1167

    PubMed Central  PubMed  CAS  Google Scholar 

  36. DePas WH, Chapman MR (2012) Microbial manipulation of the amyloid fold. Res Microbiol 163:592–606

    PubMed Central  PubMed  CAS  Google Scholar 

  37. Schwartz K, Boles BR (2013) Microbial amyloids − functions and interactions within the host. Curr Opin Microbiol 16:93–99

    PubMed Central  PubMed  CAS  Google Scholar 

  38. Larsen P, Nielsen JL, Dueholm MS et al (2007) Amyloid adhesins are abundant in natural biofilms. Environ Microbiol 9:3077–3090

    PubMed  CAS  Google Scholar 

  39. Gebbink MFBG, Claessen D, Bouma B et al (2005) Amyloids: a functional coat for microorganisms. Nat Rev Microbiol 3:333–341

    PubMed  CAS  Google Scholar 

  40. Jordal PB, Dueholm MS, Larsen P et al (2009) Widespread abundance of functional bacterial amyloid in mycolata and other gram-positive bacteria. Appl Environ Microbiol 75:4101–4110

    PubMed Central  PubMed  CAS  Google Scholar 

  41. Dueholm MS, Petersen SV, Soenderkaer M et al (2010) Functional amyloid in Pseudomonas. Mol Microbiol 77:1009–1020

    CAS  Google Scholar 

  42. Blanco LP, Evans ML, Smith DR et al (2012) Diversity, biogenesis and function of microbial amyloids. Trends Microbiol 20:66–73

    PubMed Central  PubMed  CAS  Google Scholar 

  43. Oli MW, Otoo HN, Crowley PJ et al (2012) Functional amyloid formation by Streptococcus mutans. Microbiology 158:2903–2916

    PubMed  CAS  Google Scholar 

  44. Krebs MRH, Bromley EHC, Donald AM (2005) The binding of thioflavin-T to amyloid fibrils: localisation and implications. J Struct Biol 149:30–37

    PubMed  CAS  Google Scholar 

  45. O’Nuallain B, Wetzel R (2002) Conformational Abs recognizing a generic amyloid fibril epitope. Proc Natl Acad Sci U S A 99:1485–1490

    PubMed Central  PubMed  Google Scholar 

  46. Kloecke FO, Geesey GG (1999) Localization and identification of populations of phosphatase: active bacterial cells associated with activated sludge flocs. Microb Ecol 38:201–214

    Google Scholar 

  47. Espeland EM, Wetzel RG (2001) Effects of photosynthesis on bacterial phosphatase production in biofilms. Microb Ecol 42:328–337

    PubMed  CAS  Google Scholar 

  48. Gonzalez-Gil S, Kaefer BA, Jovine RVM et al (1998) Detection and quantification of alkaline phosphatase in single cells of phosphorous-starved marine phytoplankton. Mar Ecol Prog Ser 164:21–35

    CAS  Google Scholar 

  49. Rengefors K, Pettersson K, Blenckner T, Anderson DM (2001) Species-specific alkaline phosphatase activity in freshwater spring phytoplankton: application of a novel method. J Plankton Res 23:435–443

    CAS  Google Scholar 

  50. Nedoma J, Strojsova A, Vrba J et al (2003) Extracellular phosphatase activity of natural plankton studied with ELF97 phosphate: fluorescence quantification and labelling kits. Environ Microbiol 5:462–472

    PubMed  CAS  Google Scholar 

  51. Strojsova A, Vrba J, Nedoma J, Komarkova J, Znachor P (2003) Seasonal study of extracellular phosphatase expression in the phytoplankton of a eutrophic reservoir. Eur J Phycol 38:295–306

    CAS  Google Scholar 

  52. van Aarle IM, Olsson PA, Söderström B (2001) Microscopic detection of phosphatase activity of saprophytic and arbuscular mycorrhizal fungi using a fluorogenic substrate. Mycologia 93:17–24

    Google Scholar 

  53. Murakawa T (1973) Slime production by Pseudomonas aeruginosa IV. Chemical analysis of two varieties of slime produced by Pseudomonas aeruginosa. Jpn J Microbiol 17:513–520

    PubMed  CAS  Google Scholar 

  54. Palmgren R, Nielsen PH (1996) Accumulation of DNA in the extracellular matrix of activated sludge and bacterial cultures. Water Sci Technol 34:233–240

    CAS  Google Scholar 

  55. Whitchurch CB, Tolker-Nielsen T, Ragas P, Mattick JS (2002) Extracellular DNA required for bacterial biofilm formation. Science 295:1487

    PubMed  CAS  Google Scholar 

  56. Allesen-Holm M, Bundvik Barken K, Yang L et al (2006) A characterisation of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol Microbiol 59:1114–1128

    PubMed  CAS  Google Scholar 

  57. Yang L, Barken KB, Skindersoe ME et al (2007) Effects of iron on DNA release and biofilm development by Pseudomonas aeruginosa. Microbiology 153:1318–1328

    PubMed  CAS  Google Scholar 

  58. Neu TR (1996) Significance of bacterial surface-active compounds in interaction of bacteria with interfaces. Microbiol Rev 60:151–166

    PubMed Central  PubMed  CAS  Google Scholar 

  59. Rosenberg E, Ron EZ (1999) High- and low-molecular-mass microbial surfactants. Appl Microbiol Biotechnol 52:154–162

    PubMed  CAS  Google Scholar 

  60. Schooling SR, Beveridge TJ (2006) Membrane vesicles: an overlooked component of the matrices of biofilms. J Bacteriol 188:5945–5957

    PubMed Central  PubMed  CAS  Google Scholar 

  61. Tashiro Y, Ichikawa S, Shimizu M et al (2010) Variation of physiochemical properties and cell association activity of membrane vesicles with growth phase in Pseudomonas aeruginosa. Appl Environ Microbiol 76:3732–3739

    PubMed Central  PubMed  CAS  Google Scholar 

  62. Brooks SA, Leathem AJC, Schumacher U (1997) Lectin histochemistry. A concise practical handbook. Bios Scientific Publishers, Oxford

    Google Scholar 

  63. Lawrence JR, Wolfaardt GM, Korber DR (1994) Determination of diffusion coefficients in biofilms by confocal laser microscopy. Appl Environ Microbiol 60:1166–1173

    PubMed Central  PubMed  CAS  Google Scholar 

  64. Hidalgo G, Burns A, Herz E et al (2009) Functional tomographic fluorescence imaging of pH microenvironments in microbial biofilms by use of silica nanoparticle sensors. Appl Environ Microbiol 75:7426–7435

    PubMed Central  PubMed  CAS  Google Scholar 

  65. Acosta MA, Velasquez M, Williams K et al (2012) Fluorescent silica particles for monitoring oxygen levels in three-dimensional heterogeneous cellular structures. Biotechnol Bioeng 109:2663–2670

    PubMed Central  PubMed  CAS  Google Scholar 

  66. Aldeek F, Mustin C, Balan L et al (2011) Surface-engineered quantum dots for the labeling of hydrophobic microdomains in bacterial biofilms. Biomaterials 32:5459–5470

    PubMed  CAS  Google Scholar 

  67. Aldeek F, Schneider R, Fontaine-Aupart MP et al (2013) Patterned hydrophobic domains in the exopolymer matrix of Shewanella oneidensis MR-1 biofilms. Appl Environ Microbiol 79:1400–1402

    PubMed Central  PubMed  CAS  Google Scholar 

  68. Epstein AK, Pokroy B, Seminara A, Aizenberg J (2011) Bacterial biofilm shows persistent resistance to liquid wetting and gas penetration. Proc Natl Acad Sci U S A 108:995–1000

    PubMed Central  PubMed  CAS  Google Scholar 

  69. Kobayashi K, Iwano M (2012) BslA(YuaB) forms a hydrophobic layer on the surface of Bacillus subtilis biofilms. Mol Microbiol 85:51–66

    PubMed  CAS  Google Scholar 

  70. Nielsen PH, Jahn A (1999) Extraction of EPS. In: Wingender J, Neu TR, Flemming H-C (eds) Microbial extracellular polymeric substances. Springer, Berlin, pp 49–72

    Google Scholar 

  71. Gallaher TK, Wu S, Webster P, Aguilera R (2006) Identification of biofilm proteins in non-typeable Haemophilus influenzae. BMC Microbiol 6:1–9

    Google Scholar 

  72. Curtis PD, Atwood J III, Orlando R, Shimkets LJ (2007) Proteins associated with the Myxococcus xanthus extracellular matrix. J Bacteriol 189:7634–7642

    PubMed Central  PubMed  CAS  Google Scholar 

  73. Dumas E, Meunier B, Berdague J-L et al (2008) Comparative analysis of extracellular and intracellular proteomes of Listeria monocytogenes strains reveals a correlation between protein expression and serovar. Appl Environ Microbiol 74:7399–7409

    PubMed Central  PubMed  CAS  Google Scholar 

  74. Paes Leme AF, Bellato CM, Bedi G et al (2008) Effects of Sucrose on the extracellular matrix of plaque-like biofilm formed in vivo, studied by proteomic analysis. Caries Res 42:435–443

    PubMed Central  PubMed  CAS  Google Scholar 

  75. Cao B, Shi L, Brown RN et al (2011) Extracellular polymeric substances from Shewanella sp. HRCR-1 biofilms: characterization by infrared spectroscopy and proteomics. Environ Microbiol 13:1018–1031

    PubMed  CAS  Google Scholar 

  76. Ivleva N, Wagner M, Horn H, Niessner R, Haisch C (2009) Towards a nondestructive chemical characterization of biofilm matrix by Raman microscopy. Anal Bioanal Chem 393:197–206

    PubMed  CAS  Google Scholar 

  77. Wagner M, Ivleva NP, Haisch C et al (2009) Combined use of confocal laser scanning microscopy (CLSM) and Raman microscopy (RM): investigations on EPS-matrix. Water Res 43:63–76

    PubMed  CAS  Google Scholar 

  78. Böckelmann U, Janke A, Kuhn R et al (2006) Bacterial extracellular DNA forming a defined network like structure. FEMS Microbiol Lett 262:31–38

    PubMed  Google Scholar 

  79. Guiton PS, Hung CS, Kline KA et al (2009) Contribution of autolysin and sortase A during Enterococcus faecalis DNA-dependent biofilm development. Infect Immun 77:3626–3638

    PubMed Central  PubMed  CAS  Google Scholar 

  80. Jurcisek JA, Bakaletz LO (2007) Biofilms formed by nontypeable Haemophilus influenzae in vivo contain both double-stranded DNA and type IV pilin protein. J Bacteriol 189:3868–3875

    PubMed Central  PubMed  CAS  Google Scholar 

  81. Vilain S, Pretorius JM, Theron J, Brözel VS (2009) DNA as an adhesin: Bacillus cereus requires extracellular DNA to form biofilms. Appl Environ Microbiol 75:2861–2868

    PubMed Central  PubMed  CAS  Google Scholar 

  82. Mann EE, Rice KC, Boles BR et al (2009) Modulation of eDNA release and degradation affects Staphylococcus aureus biofilm maturation. PLoS One 4:e5822

    PubMed Central  PubMed  Google Scholar 

  83. Lappmann M, Claus H, van Alen T et al (2010) A dual role of extracellular DNA during biofilm formation of Neisseria meningitis. Mol Microbiol 75:1355–1371

    Google Scholar 

  84. Seper A, Fengler VHI, Roier S et al (2011) Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation. Mol Microbiol 82:1015–1037

    PubMed Central  PubMed  CAS  Google Scholar 

  85. Goedeke J, Paul K, Lassak J, Thormann KM (2011) Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1. ISME J 5:613–626

    CAS  Google Scholar 

  86. Goedeke J, Heun M, Bubendorfer S et al (2011) Roles of two Shewanella oneidensis MR-1 extracellular endonucleases. Appl Environ Microbiol 77:5342–5351

    CAS  Google Scholar 

  87. Pinchuk GE, Ammons C, Culley DE et al (2008) Utilization of DNA as a sole source of phosphorus, carbon, and energy by Shewanella spp: ecological and physiological implications for dissimilatory metal reduction. Appl Environ Microbiol 74:1198–1208

    PubMed Central  PubMed  CAS  Google Scholar 

  88. Caldwell DE, Korber DR, Lawrence JR (1992) Confocal laser scanning microscopy and digital image analysis in microbial ecology. Adv Microb Ecol 12:1–67

    CAS  Google Scholar 

  89. Gorman SP, Mawhinney WM, Adair CG (1993) Confocal laser scanning microscopy of adherent microorganisms, biofilms and surfaces. In: Denyer SP, Gorman SP, Sussman M (eds) Microbial biofilms: formation and control. Blackwell, London, pp 95–107

    Google Scholar 

  90. Manning PA (1995) Use of confocal microscopy in studying bacterial adhesion and invasion. Methods Enzymol 253:159–167

    PubMed  CAS  Google Scholar 

  91. Lawrence JR, Korber DR, Wolfaardt GM, Caldwell DE (1996) Analytical imaging and microscopy techniques. In: Hurst CJ, Knudsen GR, McInerney MJ, Stetzenbach LD, Walter MV (eds) Manual of environmental microbiology. ASM, Washington, pp 29–51

    Google Scholar 

  92. Lawrence JR, Wolfaardt G, Neu TR (1998) The study of microbial biofilms by confocal laser scanning microscopy. In: Wilkinson MHF, Shut F (eds) Digital image analysis of microbes. Wiley, Chichester, pp 431–465

    Google Scholar 

  93. Lawrence JR, Neu TR (1999) Confocal laser scanning microscopy for analysis of microbial biofilms. Methods Enzymol 310:131–144

    PubMed  CAS  Google Scholar 

  94. Palmer RJ Jr, Sternberg C (1999) Modern microcopy in biofilm research: confocal microscopy and other approaches. Curr Opin Biotechnol 10:263–268

    PubMed  CAS  Google Scholar 

  95. Adair CG, Gorman SP, Byers LB et al (2000) Confocal laser scanning microscopy for examination of microbial biofilms. In: An YH, Friedman RJ (eds) Handbook of bacterial adhesion. Humana, Totowa, pp 249–256

    Google Scholar 

  96. Neu TR, Kuhlicke U, Lawrence JR (2002) Assessment of fluorochromes for two-photon laser scanning microscopy biofilms. Appl Environ Microbiol 68:901–909

    PubMed Central  PubMed  CAS  Google Scholar 

  97. Lawrence JR, Korber DR, Wolfaardt GM et al (2002) Analytical imaging and microscopy techniques. In: Hurst CJ, Crawford RL, Knudsen GR, McInerney MJ, Stetzenbach LD (eds) Manual of environmental microbiology. ASM, Washington, pp 39–61

    Google Scholar 

  98. Li Y, Dick WA, Tuovinen OH (2004) Fluorescence microscopy for visualisation of soil microorganisms: a review. Biol Fertil Soils 39:301–311

    Google Scholar 

  99. Schmid M, Rothballer M, Assmus B et al (2004) Detection of microbes by scanning confocal laser microscopy (SCLM). In: Kowalchuk GA, Bruijn FJ, Head IM, Akkermans ADL, Elsas JD (eds) Molecular microbial ecology manual. Kluwer Academic Publishers, Dordrecht, pp 875–910

    Google Scholar 

  100. Neu TR, Lawrence JR (2005) One-photon versus two-photon laser scanning microscopy and digital image analysis of microbial biofilms. Methods Microbiol 34:87–134

    Google Scholar 

  101. Palmer RJ Jr, Haagensen J, Neu TR, Sternberg C (2006) Confocal microscopy of biofilms: spatiotemporal approaches. In: Pawley JB (ed) Handbook of biological confocal microscopy. Springer, New York, pp 882–900

    Google Scholar 

  102. Lawrence JR, Korber D, Neu TR (2007) Analytical imaging and microscopy techniques. In: Hurst CJ, Crawford RL, Garland JL, Lipson DA, Mills AL, Stetzenbach LD (eds) Manual of environmental microbiology. ASM, Washington, DC, pp 40–68

    Google Scholar 

  103. Lawrence JR, Neu TR (2007) Laser scanning microscopy. In: Reddy CA, Beveridge TJ, Breznak JA, Marzluf GA, Schmidt TM, Snyder LR (eds) Methods for general and molecular microbiology. ASM, Washington DC, pp 34–53

    Google Scholar 

  104. Lawrence JR, Neu TR (2007) Laser scanning microscopy for microbial flocs and particles. In: Wilkinson KJ, Lead JR (eds) Environmental colloids: behavior, structure and characterisation. John Wiley, Chichester, pp 469–505

    Google Scholar 

  105. Neu TR, Lawrence JR (2010) Examination of microbial communities on hydrocarbons by means of laser scanning microscopy. In: Timmis KN (ed) Microbiology of hydrocarbons, oils, lipids and derived compounds. Springer, Heidelberg, pp 4073–4084

    Google Scholar 

  106. Neu TR, Manz B, Volke F et al (2010) Advanced imaging techniques for assessment of structure, composition and function in biofilm systems. FEMS Microbiol Ecol 72:1–21

    PubMed  CAS  Google Scholar 

  107. Brown MJ, Lester JN (1980) Comparison of bacterial extracellular polymer extraction methods. Appl Environ Microbiol 40:179–185

    PubMed Central  PubMed  CAS  Google Scholar 

  108. Jahn A, Nielsen PH (1995) Extraction of extracellular polymeric substances (EPS) from biofilms using a cation exchange resin. Water Sci Technol 32:157–164

    CAS  Google Scholar 

  109. Zhang X, Bishop PL, Kinkle BK (1999) Comparison of extraction methods for quantifying extracellular polymers in biofilms. Water Sci Technol 39:211–218

    CAS  Google Scholar 

  110. Liu H, Fang HHP (2002) Extraction of extracellular polymeric substances (EPS) of sludges. J Biotechnol 95:249–256

    PubMed  CAS  Google Scholar 

  111. Comte S, Guibaud G, Baudu M (2006) Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: part I. Comparison of the efficiency of eight EPS extraction methods. Enzyme Microb Technol 38:237–245

    CAS  Google Scholar 

  112. Klock J-H, Wieland A, Seifert R, Michaelis W (2007) Extracellular polymeric substances (EPS) from cyanobacterial mats: characterisation and isolation method optimisation. Marine Biol 152:1077–1085

    CAS  Google Scholar 

  113. Ras M, Girbal-Neuhauser E, Paul E et al (2008) Protein extraction from activated sludge: an analytical approach. Water Res 42:1867–1878

    PubMed  CAS  Google Scholar 

  114. Aguilera A, Souza-Egipsy V, San Martin-Uriz P, Amils R (2008) Extraction of extracellular polymeric substances from extreme acidic microbial biofilms. Appl Microbiol Biotechnol 78:1079–1088

    PubMed  CAS  Google Scholar 

  115. Wu J, Xi C (2009) Evaluation of different methods for extracting extracellular DNA from the biofilm matrix. Appl Environ Microbiol 75:5390–5395

    PubMed Central  PubMed  CAS  Google Scholar 

  116. D’Abzac P, Bordas F, van Hullebusch E et al (2010) Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludge: comparison of chemical and physical extraction protocols. Appl Microbiol Biotechnol 85:1589–1599

    PubMed  Google Scholar 

  117. Wei LL, Wang K, Zhao QL et al (2012) Fractional, biodegradable and spectral characteristics of extracted and fractionated sludge extracellular polymeric substances. Water Res 46:4387–4396

    PubMed  CAS  Google Scholar 

  118. Sun M, Li WW, Yu HQ, Harada H (2012) A novel integrated approach to quantitatively evaluate the efficiency of extracellular polymeric substances (EPS) extraction process. Appl Microbiol Biotechnol 96:1577–1585

    PubMed  CAS  Google Scholar 

  119. Zhang L, Ren H, Ding L (2012) Comparison of extracellular polymeric substances (EPS) extraction from two different activated sludges. Water Sci Technol 66:1558–1564

    PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. UFZ, Helmholtz Centre for Environmental Research, Brueckstrasse 3A, 39114, Magdeburg, Germany

    Thomas R. Neu

  2. Environment Canada, Saskatoon, SK, Canada

    John R. Lawrence

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  1. Thomas R. Neu
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  2. John R. Lawrence
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Correspondence to Thomas R. Neu .

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  1. Fondazione Santa Lucia IRCCS Microbial Biofilm Laboratory, Rome, Italy

    Gianfranco Donelli

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Neu, T.R., Lawrence, J.R. (2014). Advanced Techniques for In Situ Analysis of the Biofilm Matrix (Structure, Composition, Dynamics) by Means of Laser Scanning Microscopy. In: Donelli, G. (eds) Microbial Biofilms. Methods in Molecular Biology, vol 1147. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0467-9_4

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  • DOI: https://doi.org/10.1007/978-1-4939-0467-9_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0466-2

  • Online ISBN: 978-1-4939-0467-9

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