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List of Contributors xii <p>Introduction xvi</p> <p>Guide to Methods xviii</p> <p><b>Part I Methods for Microfouling 1</b></p> <p>Part Editor: Sergey Dobretsov</p> <p><b>1 Microscopy of biofilms 3</b></p> <p><b>Section 1 Traditional light and epifluorescent microscopy 4</b><br /> <i>Sergey Dobretsov and Raeid M.M. Abed</i></p> <p>1.1 Introduction 4</p> <p>1.2 Determination of bacterial abundance 8</p> <p>1.3 Catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH) 9</p> <p>1.4 Suggestions, with examples, for data analysis and presentation 12</p> <p>Acknowledgements 13</p> <p>References 13</p> <p><b>Section 2 Confocal laser scanning microscopy 15</b><br /> <i>Koty Sharp</i></p> <p>1.5 Introduction 15</p> <p>1.6 Materials, equipment, and method 18</p> <p>1.7 Image acquisition 21</p> <p>1.8 Presentation 21</p> <p>1.9 Troubleshooting hints and tips 21</p> <p>1.10 Notes 23</p> <p>References 23</p> <p><b>Section 3 Electron microscopy 26</b><br /> <i>Omar Skalli, Lou G. Boykins, and Lewis Coons</i></p> <p>1.11 Introduction 26</p> <p>1.12 Transmission electron microscopy (TEM) 27</p> <p>1.13 Scanning electron microscopy (SEM) 35</p> <p>References 40</p> <p><b>2 Traditional and bulk methods for biofilms 44</b></p> <p><b>Section 1 Traditional microbiological methods 45</b><br /> <i>Hans-Uwe Dahms</i></p> <p>2.1 Introduction 45</p> <p>2.2 Enrichment culture, isolation of microbes 45</p> <p>2.3 Counting methods 48</p> <p>2.4 Troubleshooting hints and tips 49</p> <p>References 50</p> <p><b>Section 2 Bulk methods 52</b><br /> <i>Sergey Dobretsov</i></p> <p>2.5 Introduction 52</p> <p>2.6 Measurement of biofilm thickness 53</p> <p>2.7 Biofilm dry weight determination 54</p> <p>2.8 Biofilm ATP content 55</p> <p>2.9 Troubleshooting hints and tips 56</p> <p>Acknowledgements 57</p> <p>References 57</p> <p><b>3 Biocide testing against microbes 58</b></p> <p><b>Section 1 Testing biocides in solution: flow cytometry for planktonic stages 59</b><br /> <i>Tristan Biggs, Tom Vance, and Glen Tarran</i></p> <p>3.1 Introduction 59</p> <p>3.2 Method introductions 60</p> <p>3.3 Pros and cons 66</p> <p>3.4 Materials and equipment 67</p> <p>3.5 Methods 68</p> <p>3.6 Troubleshooting hints and tips 70</p> <p>3.7 Suggestions 71</p> <p>References 72</p> <p><b>Section 2 Biocide testing using single and multispecies biofilms 76</b><br /> <i>Torben Lund Skovhus</i></p> <p>3.8 Introduction 76</p> <p>3.9 Questions to answer when applying biocides 76</p> <p>3.10 Laboratory methods for testing biocide effect 78</p> <p>3.11 Field methods for testing biocide effect 81</p> <p>3.12 Troubleshooting hints and tips 83</p> <p>Acknowledgements 84</p> <p>References 84</p> <p><b>4 Molecular methods for biofilms 87</b></p> <p><b>Section 1 Isolation of nucleic acids 88</b><br /> <i>Isabel Ferrera and Vanessa Balagué</i></p> <p>4.1 Introduction 88</p> <p>4.2 Materials 89</p> <p>4.3 Isolation of DNA from a biofilm 90</p> <p>4.4 Troubleshooting hints and tips 91</p> <p>References 91</p> <p><b>Section 2 PCR and DNA sequencing 93</b><br /> <i>Christian R. Voolstra, Manuel Aranda, and Till Bayer</i></p> <p>4.5 PCR and DNA sequencing: General introduction 93</p> <p>4.6 PCR 93</p> <p>4.7 Microbial marker genes – 16S 94</p> <p>4.8 DNA sequencing 95</p> <p>4.9 454 16S amplicon pyrotag sequencing 95</p> <p>4.10 Protocol 1: DNA extraction using the Qiagen DNeasy Plant Mini Kit 96</p> <p>4.11 Protocol 2: Full-length 16S PCR using the Qiagen Multiplex Kit 98</p> <p>4.12 Protocol 3: Analysis of full-length 16S genes 100</p> <p>4.13 Protocol 4: 16S amplicon PCR for 454 sequencing using the Qiagen Multiplex Kit 102</p> <p>4.14 Protocol 5: Trimming and filtering of 454 16S pyrotag sequencing 106</p> <p>4.15 Protocol 6: Taxon-based analyses 108</p> <p>4.16 Protocol 7: Phylogeny-based analyses 109</p> <p>References 111</p> <p><b>Section 3 Community comparison by genetic fingerprinting techniques 114</b><br /> <i>Raeid M.M. Abed and Sergey Dobretsov</i></p> <p>4.17 Introduction 114</p> <p>4.18 History and principles of the methods 115</p> <p>4.19 Advantages and limitations of fingerprinting techniques 116</p> <p>4.20 Materials and equipment 116</p> <p>4.21 Suggestions for data analysis and presentation 121</p> <p>4.22 Troubleshooting hints and tips 121</p> <p>Acknowledgements 122</p> <p>References 122</p> <p><b>Section 4 Metagenomics 125</b><br /> <i>Sarah M. Owens, Jared Wilkening, Jennifer L. Fessle, and Jack A. Gilbert</i></p> <p>4.23 Introduction and brief summary of methods 125</p> <p>4.24 Overview of metagenomics methods 125</p> <p>4.25 Method introduction 126</p> <p>4.26 Overview of DNA handling for BAC library construction 127</p> <p>4.27 BAC and Fosmid library construction 127</p> <p>4.28 Library handling, archiving, and databasing 128</p> <p>4.29 Facilitating library screening 128</p> <p>4.30 Time frame considerations 129</p> <p>4.31 Materials and equipment 129</p> <p>4.32 Detailed methods: DNA handling and BAC library construction 130</p> <p>4.33 Troubleshooting tips 131</p> <p>4.34 Suggestions for data analysis 132</p> <p>4.35 Suggestions for presentation of data 134</p> <p>Acknowledgements 135</p> <p>References 135</p> <p><b>5 Methods for biofilm constituents and turnover 138</b></p> <p><b>Section 1 Destructive and nondestructive methods 139</b><br /> <i>Arnaud Bridier, Florence Dubois-Brissonnet, and Romain Briandet</i></p> <p>5.1 Introduction 139</p> <p>5.2 Pros and cons of destructive and nondestructive M-LSM methods for biofilm analysis 140</p> <p>5.3 Materials and equipment required for M-LSM 140</p> <p>5.4 Example of questions than can be answered with the method 140</p> <p>5.5 Suggestions for data analysis and presentation 148</p> <p>References 149</p> <p><b>Section 2 Biofilm formation and quorum sensing bioassays 153</b><br /> <i>Clayton E. Cox, William J. Zaragoza, Cory J. Krediet, and Max Teplitski</i></p> <p>5.6 Introduction 153</p> <p>5.7 Materials and equipment 157</p> <p>5.8 Methods 157</p> <p>Acknowledgements 165</p> <p>References 165</p> <p><b>6 Sampling and experiments with biofilms in the environment 168</b></p> <p><b>Section 1 Field trials with biofilms 169</b><br /> <i>Jeremy C. Thomason</i></p> <p>6.1 Introduction 169</p> <p>6.2 Materials and equipment 170</p> <p>6.3 Method 170</p> <p>6.4 Troubleshooting hints and tips 171</p> <p>6.5 Suggestions for data analysis and presentation 172</p> <p>References 173</p> <p><b>Section 2 Sampling from large structures such as ballast tanks 175</b><br /> <i>Robert L. Forsberg, Anne E. Meyer, and Robert E. Baier</i></p> <p>6.6 Introduction 175</p> <p>6.7 Materials and equipment 178</p> <p>6.8 Troubleshooting hints and tips 180</p> <p>6.9 Analytical methods 180</p> <p>6.10 Suggestions for data analysis and presentation 182</p> <p>References 182</p> <p><b>Section 3 Sampling from living organisms 184</b><br /> <i>Christina A. Kellogg</i></p> <p>6.11 Introduction 184</p> <p>6.12 Historical background 185</p> <p>6.13 Advantages and limitations of collection techniques 185</p> <p>6.14 Protocols 186</p> <p>6.15 Suggestions for data analysis 187</p> <p>6.16 Troubleshooting hints and tips 187</p> <p>Acknowledgment 188</p> <p>References 188</p> <p><b>Section 4 Optical methods in the field 190</b><br /> <i>Richard J. Murphy</i></p> <p>6.17 Introduction 190</p> <p>6.18 Examples of the use of optical methods 191</p> <p>6.19 Spectral characteristics of biofilms 192</p> <p>6.20 The use of chlorophyll-a as an index of biomass of biofilm 193</p> <p>6.21 Multi-versus hyperspectral measurements (CIR imagery versus field spectrometry) 194</p> <p>6.22 Calibration of data to reflectance 195</p> <p>6.23 Suggestions for data analysis and presentation 195</p> <p>6.24 Methods 197</p> <p>6.25 Troubleshooting hints and tips 201</p> <p>References 202</p> <p><b>7 Laboratory experiments and cultures 204</b></p> <p><b>Section 1 Static, constant depth and/or flow cells 205</b><br /> <i>Robert L. Forsberg, Anne E. Meyer, and Robert E. Baier</i></p> <p>7.1 Introduction 205</p> <p>7.2 Portable Biofouling Unit 207</p> <p>7.3 Pros and cons of the method 207</p> <p>7.4 Materials and equipment 208</p> <p>7.5 Suggestions for data analysis 209</p> <p>7.6 “Benchmark” bacteria and biofilm characterization 210</p> <p>7.7 Troubleshooting hints and tips 212</p> <p>References 212</p> <p><b>Section 2 Mixed population fermentor 214</b><br /> <i>Jennifer Longyear</i></p> <p>7.8 Introduction 214</p> <p>7.9 Pros and cons 215</p> <p>7.10 Fermentor 215</p> <p>7.11 Mixed species microfouling culture 215</p> <p>7.12 Utilizing the fermentor test section 218</p> <p>7.13 Troubleshooting, hints and tips 218</p> <p>References 219</p> <p><b>Part II Methods for Macrofouling, Coatings and Biocides 221</b></p> <p>Part Editors: Jeremy C. Thomason, David N. Williams.</p> <p><b>8 Measuring larval availability, supply and behavior 223</b></p> <p><b>Section 1 Larval availability and supply 224</b><br /> <i>Sarah Dudas and Joe Tyburczy</i></p> <p>8.1 Introduction to measuring larval availability and supply 224</p> <p>8.2 Measuring settlement and recruitment 235</p> <p>References 238</p> <p><b>Section 2 Larval behavior 241</b><br /> <i>Jeremy C. Thomason</i></p> <p>8.3 Introduction 241</p> <p>8.4 Method for tracking larvae 242</p> <p>8.5 Troubleshooting hints and tips 245</p> <p>8.6 Suggestions for data analysis and presentation 246</p> <p>References 249</p> <p><b>9 Assessing macrofouling 251</b></p> <p><b>Section 1: Assessing fouling assemblages 252</b><br /> <i>João Canning-Clode and Heather Sugden</i></p> <p>9.1 Introduction 252</p> <p>9.2 A note on taxonomy 253</p> <p>9.3 Field methods 253</p> <p>9.4 Digital methods 258</p> <p>9.5 Functional groups 261</p> <p>9.6 Predicting total richness: from the known to the unknown 264</p> <p>References 267</p> <p><b>Section 2 Assessment of in-service vessels for biosecurity risk 271</b><br /> <i>Francisco Sylvester and Oliver Floerl</i></p> <p>9.7 Introduction 271</p> <p>9.8 Surveys of vessel hulls 272</p> <p>9.9 Sample and data analysis 277</p> <p>Acknowledgements 279</p> <p>References 279</p> <p><b>Section 3 Experiments on a global scale 281</b><br /> <i>Mark Lenz</i></p> <p>9.10 Experiments in ecology: the need for scaling up 281</p> <p>9.11 GAME – a program for modular experimental research in marine ecology 281</p> <p>9.12 Marine macrofouling communities as model systems 282</p> <p>9.13 Chronology of a GAME project 283</p> <p>Acknowledgements 289</p> <p>References 289</p> <p><b>10 Efficacy testing of nonbiocidal and fouling-release coatings 291</b><br /> <i>Maureen E. Callow, James A. Callow, Sheelagh Conlan, Anthony S. Clare, and Shane Stafslien</i></p> <p>10.1 Introduction 291</p> <p>10.2 Test organisms 293</p> <p>10.3 Test samples 294</p> <p>10.4 “Antifouling” settlement assays 295</p> <p>10.5 Fouling-release assays 299</p> <p>10.6 Adhesion assays for high-throughput screening 304</p> <p>10.7 Apparatus 310</p> <p>Acknowledgements 313</p> <p>References 314</p> <p><b>11 Contact angle measurements 317</b></p> <p><b>Section 1 Surface characterization by contact angle measurements 318</b><br /> <i>Doris M. Fopp-Spori</i></p> <p>11.1 Introduction 318</p> <p>11.2 Liquids in contact with solids 318</p> <p>11.3 Reproducible contact angle measurements 320</p> <p>11.4 Surface energy calculations 323</p> <p>References 324</p> <p><b>Section 2 Underwater contact angle measurement by the captive bubble method 326<br /> </b><i>Pierre Martin-Tanchereau</i></p> <p>11.5 Introduction 326</p> <p>11.6 Materials and requirements 327</p> <p>11.7 Method 329</p> <p>11.8 Surface energy 330</p> <p>Acknowledgements 330</p> <p>References 331</p> <p><b>12 Efficacy testing of biocides and biocidal coatings</b> <b>332</b><br /> <i>Christine Bressy, Jean-François Briand, Chantal Compère, and Karine Réhel</i></p> <p>12.1 Introduction 332</p> <p>12.2 Laboratory assays for biocides 333</p> <p>12.3 Field test methodology for biocidal coatings 337</p> <p>References 343</p> <p>13 Commercialization 346</p> <p><b>Section 1 Processing a new marine biocide from innovation through regulatory approvals towards commercialization 347</b><br /> <i>Lena Lindblat</i></p> <p>13.1 Introduction 347</p> <p>13.2 Basics about the regulatory landscape from the academic perspective 349</p> <p>13.3 Risk, risk assessment and risk management 349</p> <p>13.4 Future directions 353</p> <p>13.5 Conclusions 355</p> <p>References 356</p> <p><b>Section 2 From laboratory to ship: pragmatic development of fouling control coatings in industry 358</b><br /> <i>Richie Ramsden and Jennifer Longyear</i></p> <p>13.6 Introduction 358</p> <p>13.7 Laboratory coating development 358</p> <p>13.8 Laboratory bioassay screening 359</p> <p>13.9 Fitness for purpose (FFP) testing 360</p> <p>13.10 Field antifouling performance testing 361</p> <p>13.11 Test patch and vessel trials 363</p> <p>13.12 Performance monitoring 364</p> <p>13.13 Summary 365</p> <p>References 365</p> <p>Index 366</p>

<p><b>Dr. Sergey Dobretsov</b> has worked for more than 20 years on biofouling, is widely published, and is the co-inventor on four international antifouling patents. He trained as a biologist in St Petersburg State University, Russia, and has worked in leading biofouling research centers in Russia, Hong Kong, Germany, and the USA. He is currently an Assistant Professor at Sultan Qaboos University, Oman. He is on the editorial boards of the journals <i>Marine Ecology Progress Series</i> and <i>Biofouling</i>.</p> <p><b>Dr. David N. Williams</b> is the RD&I Director for AkzoNobel Marine & Protective Coatings. Based in the North East of England he originally trained as a chemist at Durham University and at Lausanne University, Switzerland. His specific expertise is in the area of nonbiocidal antifouling technologies and he is the co-inventor on a number of patents on silicone foul-release coatings and applications.</p> <p><b>Dr. Jeremy C. Thomason</b> is a marine biologist, a former academic at a British University and Royal Society Industrial Research Fellow, and now runs a scientific and technical consultancy, Ecoteknica, from the Yucatán, México. He has worked in the field of biofouling for more than 20 years, is co-inventor on several patents, and is a co-editor of the book <i>Biofouling</i> also published by Wiley Blackwell in 2010.</p>

<p><i>Biofouling Methods</i> provides a “cook book” for both established workers and those new to the field. The methods included in this important new book range from tried and tested techniques to those at the cutting edge, encompassing the full diversity of this multidisciplinary field.</p> <p>The book covers methods for microbial and macrofouling, coatings and biocides, and ranges from methods for fundamental studies to methods relevant for industrial applications. There is an emphasis on answering questions and each chapter provides technical methods and problem-solving hints and tips.</p> <p>Bringing together a wealth of international contributions and edited by three internationally known and respected experts in the subject, <i>Biofouling Methods</i> is the essential methodology reference in the field for all those working in the antifouling industry, including those involved in formulation of antifouling products such as paints and other coatings. Aquatic biologists, ecologists, environmental scientists and lawyers, marine engineers, aquaculture personnel, chemists, and medical researchers will all find much of interest within this book. All universities and research establishments where these subjects are studied and taught should have copies of this important work on their shelves.</p>

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