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Introduction xvi
Guide to Methods xviii
Part I Methods for Microfouling 1
Part Editor: Sergey Dobretsov
1 Microscopy of biofilms 3
Section 1 Traditional light and epifluorescent microscopy 4Sergey Dobretsov and Raeid M.M. Abed
1.1 Introduction 4
1.2 Determination of bacterial abundance 8
1.3 Catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH) 9
1.4 Suggestions, with examples, for data analysis and presentation 12
Acknowledgements 13
References 13
Section 2 Confocal laser scanning microscopy 15Koty Sharp
1.5 Introduction 15
1.6 Materials, equipment, and method 18
1.7 Image acquisition 21
1.8 Presentation 21
1.9 Troubleshooting hints and tips 21
1.10 Notes 23
References 23
Section 3 Electron microscopy 26Omar Skalli, Lou G. Boykins, and Lewis Coons
1.11 Introduction 26
1.12 Transmission electron microscopy (TEM) 27
1.13 Scanning electron microscopy (SEM) 35
References 40
2 Traditional and bulk methods for biofilms 44
Section 1 Traditional microbiological methods 45Hans-Uwe Dahms
2.1 Introduction 45
2.2 Enrichment culture, isolation of microbes 45
2.3 Counting methods 48
2.4 Troubleshooting hints and tips 49
References 50
Section 2 Bulk methods 52Sergey Dobretsov
2.5 Introduction 52
2.6 Measurement of biofilm thickness 53
2.7 Biofilm dry weight determination 54
2.8 Biofilm ATP content 55
2.9 Troubleshooting hints and tips 56
Acknowledgements 57
References 57
3 Biocide testing against microbes 58
Section 1 Testing biocides in solution: flow cytometry for planktonic stages 59Tristan Biggs, Tom Vance, and Glen Tarran
3.1 Introduction 59
3.2 Method introductions 60
3.3 Pros and cons 66
3.4 Materials and equipment 67
3.5 Methods 68
3.6 Troubleshooting hints and tips 70
3.7 Suggestions 71
References 72
Section 2 Biocide testing using single and multispecies biofilms 76Torben Lund Skovhus
3.8 Introduction 76
3.9 Questions to answer when applying biocides 76
3.10 Laboratory methods for testing biocide effect 78
3.11 Field methods for testing biocide effect 81
3.12 Troubleshooting hints and tips 83
Acknowledgements 84
References 84
4 Molecular methods for biofilms 87
Section 1 Isolation of nucleic acids 88Isabel Ferrera and Vanessa Balagué
4.1 Introduction 88
4.2 Materials 89
4.3 Isolation of DNA from a biofilm 90
4.4 Troubleshooting hints and tips 91
References 91
Section 2 PCR and DNA sequencing 93Christian R. Voolstra, Manuel Aranda, and Till Bayer
4.5 PCR and DNA sequencing: General introduction 93
4.6 PCR 93
4.7 Microbial marker genes - 16S 94
4.8 DNA sequencing 95
4.9 454 16S amplicon pyrotag sequencing 95
4.10 Protocol 1: DNA extraction using the Qiagen DNeasy Plant Mini Kit 96
4.11 Protocol 2: Full-length 16S PCR using the Qiagen Multiplex Kit 98
4.12 Protocol 3: Analysis of full-length 16S genes 100
4.13 Protocol 4: 16S amplicon PCR for 454 sequencing using the Qiagen Multiplex Kit 102
4.14 Protocol 5: Trimming and filtering of 454 16S pyrotag sequencing 106
4.15 Protocol 6: Taxon-based analyses 108
4.16 Protocol 7: Phylogeny-based analyses 109
References 111
Section 3 Community comparison by genetic fingerprinting techniques 114Raeid M.M. Abed and Sergey Dobretsov
4.17 Introduction 114
4.18 History and principles of the methods 115
4.19 Advantages and limitations of fingerprinting techniques 116
4.20 Materials and equipment 116
4.21 Suggestions for data analysis and presentation 121
4.22 Troubleshooting hints and tips 121
Acknowledgements 122
References 122
Section 4 Metagenomics 125Sarah M. Owens, Jared Wilkening, Jennifer L. Fessle, and Jack A. Gilbert
4.23 Introduction and brief summary of methods 125
4.24 Overview of metagenomics methods 125
4.25 Method introduction 126
4.26 Overview of DNA handling for BAC library construction 127
4.27 BAC and Fosmid library construction 127
4.28 Library handling, archiving, and databasing 128
4.29 Facilitating library screening 128
4.30 Time frame considerations 129
4.31 Materials and equipment 129
4.32 Detailed methods: DNA handling and BAC library construction 130
4.33 Troubleshooting tips 131
4.34 Suggestions for data analysis 132
4.35 Suggestions for presentation of data 134
Acknowledgements 135
References 135
5 Methods for biofilm constituents and turnover 138
Section 1 Destructive and nondestructive methods 139Arnaud Bridier, Florence Dubois-Brissonnet, and Romain Briandet
5.1 Introduction 139
5.2 Pros and cons of destructive and nondestructive M-LSM methods for biofilm analysis 140
5.3 Materials and equipment required for M-LSM 140
5.4 Example of questions than can be answered with the method 140
5.5 Suggestions for data analysis and presentation 148
References 149
Section 2 Biofilm formation and quorum sensing bioassays 153Clayton E. Cox, William J. Zaragoza, Cory J. Krediet, and Max Teplitski
5.6 Introduction 153
5.7 Materials and equipment 157
5.8 Methods 157
Acknowledgements 165
References 165
6 Sampling and experiments with biofilms in the environment 168
Section 1 Field trials with biofilms 169Jeremy C. Thomason
6.1 Introduction 169
6.2 Materials and equipment 170
6.3 Method 170
6.4 Troubleshooting hints and tips 171
6.5 Suggestions for data analysis and presentation 172
References 173
Section 2 Sampling from large structures such as ballast tanks 175Robert L. Forsberg, Anne E. Meyer, and Robert E. Baier
6.6 Introduction 175
6.7 Materials and equipment 178
6.8 Troubleshooting hints and tips 180
6.9 Analytical methods 180
6.10 Suggestions for data analysis and presentation 182
References 182
Section 3 Sampling from living organisms 184Christina A. Kellogg
6.11 Introduction 184
6.12 Historical background 185
6.13 Advantages and limitations of collection techniques 185
6.14 Protocols 186
6.15 Suggestions for data analysis 187
6.16 Troubleshooting hints and tips 187
Acknowledgment 188
References 188
Section 4 Optical methods in the field 190Richard J. Murphy
6.17 Introduction 190
6.18 Examples of the use of optical methods 191
6.19 Spectral characteristics of biofilms 192
6.20 The use of chlorophyll-a as an index of biomass of biofilm 193
6.21 Multi-versus hyperspectral measurements (CIR imagery versus field spectrometry) 194
6.22 Calibration of data to reflectance 195
6.23 Suggestions for data analysis and presentation 195
6.24 Methods 197
6.25 Troubleshooting hints and tips 201
References 202
7 Laboratory experiments and cultures 204
Section 1 Static, constant depth and/or flow cells 205Robert L. Forsberg, Anne E. Meyer, and Robert E. Baier
7.1 Introduction 205
7.2 Portable Biofouling Unit 207
7.3 Pros and cons of the method 207
7.4 Materials and equipment 208
7.5 Suggestions for data analysis 209
7.6 "Benchmark" bacteria and biofilm characterization 210
7.7 Troubleshooting hints and tips 212
References 212
Section 2 Mixed population fermentor 214Jennifer Longyear
7.8 Introduction 214
7.9 Pros and cons 215
7.10 Fermentor 215
7.11 Mixed species microfouling culture 215
7.12 Utilizing the fermentor test section 218
7.13 Troubleshooting, hints and tips 218
References 219
Part II Methods for Macrofouling, Coatings and Biocides 221
Part Editors: Jeremy C. Thomason, David N. Williams.
8 Measuring larval availability, supply and behavior 223
Section 1 Larval availability and supply 224Sarah Dudas and Joe Tyburczy
8.1 Introduction to measuring larval availability and supply 224
8.2 Measuring settlement and recruitment 235
References 238
Section 2 Larval behavior 241Jeremy C. Thomason
8.3 Introduction 241
8.4 Method for tracking larvae 242
8.5 Troubleshooting hints and tips 245
8.6 Suggestions for data analysis and presentation 246
References 249
9 Assessing macrofouling 251
Section 1: Assessing fouling assemblages 252João Canning-Clode and Heather Sugden
9.1 Introduction 252
9.2 A note on taxonomy 253
9.3 Field methods 253
9.4 Digital methods 258
9.5 Functional groups 261
9.6 Predicting total richness: from the known to the unknown 264
References 267
Section 2 Assessment of in-service vessels for biosecurity risk 271Francisco Sylvester and Oliver Floerl
9.7 Introduction 271
9.8 Surveys of vessel hulls 272
9.9 Sample and data analysis 277
Acknowledgements 279
References 279
Section 3 Experiments on a global scale 281Mark Lenz
9.10 Experiments in ecology: the need for scaling up 281
9.11 GAME - a program for modular experimental research in marine ecology 281
9.12 Marine macrofouling communities as model systems 282
9.13 Chronology of a GAME project 283
Acknowledgements 289
References 289
10 Efficacy testing of nonbiocidal and fouling-release coatings 291Maureen E. Callow, James A. Callow, Sheelagh Conlan, Anthony S. Clare, and Shane Stafslien
10.1 Introduction 291
10.2 Test organisms 293
10.3 Test samples 294
10.4 "Antifouling" settlement assays 295
10.5 Fouling-release assays 299
10.6 Adhesion assays for high-throughput screening 304
10.7 Apparatus 310
Acknowledgements 313
References 314
11 Contact angle measurements 317
Section 1 Surface characterization by contact angle measurements 318Doris M. Fopp-Spori
11.1 Introduction 318
11.2 Liquids in contact with solids 318
11.3 Reproducible contact angle measurements 320
11.4 Surface energy calculations 323
References 324
Section 2 Underwater contact angle measurement by the captive bubble method 326Pierre Martin-Tanchereau
11.5 Introduction 326
11.6 Materials and requirements 327
11.7 Method 329
11.8 Surface energy 330
Acknowledgements 330
References 331
12 Efficacy testing of biocides and biocidal coatings 332Christine Bressy, Jean-François Briand, Chantal Compère, and Karine Réhel
12.1 Introduction 332
12.2 Laboratory assays for biocides 333
12.3 Field test methodology for biocidal coatings 337
References 343
13 Commercialization 346
Section 1 Processing a new marine biocide from innovation through regulatory approvals towards commercialization 347Lena Lindblat
13.1 Introduction 347
13.2 Basics about the regulatory landscape from the academic perspective 349
13.3 Risk, risk assessment and risk management 349
13.4 Future directions 353
13.5 Conclusions 355
References 356
Section 2 From laboratory to ship: pragmatic development of fouling control coatings in industry 358Richie Ramsden and Jennifer Longyear
13.6 Introduction 358
13.7 Laboratory coating development 358
13.8 Laboratory bioassay screening 359
13.9 Fitness for purpose (FFP) testing 360
13.10 Field antifouling performance testing 361
13.11 Test patch and vessel trials 363
13.12 Performance monitoring 364
13.13 Summary 365
References 365
Index 366
Sergey Dobretsov1 and Raeid M.M. Abed2
1 Department of Marine Science and Fisheries, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al Khoud, Muscat, Oman
2 Biology Department, College of Science, Sultan Qaboos University, Al Khoud, Muscat, Oman
Light microscopy is among the oldest methods used to investigate microorganisms [1, 2]. Early microscopic observations are usually associated with the name of Antony van Leeuwenhoek, who was able to magnify microorganisms 200 times using his designed microscope [1]. A modern light microscope has a magnification of about 1000× and is able to resolve objects separated by 0.275 μm. This resolving power is limited by the wavelength of the used light for the illumination of the specimens. Several light microscopy techniques, such as bright field, dark field and phase contrast, enhance contrast between microorganisms and background [1]. Fluorescent microscopy takes advantage of the ability of some materials or organisms to emit visible light when irradiated with ultraviolet radiation at a specific wavelength. Phototrophic organisms have a natural fluorescence due to the presence of chlorophyll in their cells [3]. Other organisms require additional dyes in order to become fluorescent.
Light microscopy is a simple and cheap method [2]. It is commonly used for observation of relatively large (>0.5 μm) cells of microorganisms (Figure 1.1). In comparison, epifluorescent microscopy provides higher resolution and is generally used for observation of bacteria or cell organelles. The pros and cons of these methods are presented in Table 1.1.
Figure 1.1 Microfouling community dominated by different cyanobacteria, diatoms and bacteria under a light microscope. Magnification 100×. Picture by Julie Piraino.
Table 1.1 Pros and cons of light and epifluorescent microscopy.
Epifluorescent stains allow quick and automatic counting of bacteria using flow cytometry (discussed later in this chapter). Epifluorescent microscopy is preferable over scanning electron microscopy (SEM) (Chapter 1, section 3) for bacterial size and abundance studies [4]. While direct light microscopy measurements can be highly sensitive to low cell numbers, electron microscopy methods are not. Light and epifluorescent microscopy has the advantage over electron microscopy that a larger surface area can be assessed for a given amount of time [5]. Two fluorescent stains are widely used to stain microbial cells, namely 4',6-diamidino-2-phenylindole (DAPI), which binds to DNA [6] (Figure 1.2), and acrydine orange, which binds to DNA and RNA as well as to detritus particles [7]. Therefore, the estimated number of bacteria stained with DAPI is on average 70% of bacterial counts made with acrydine orange [8]. The use of DAPI stain allows a longer period between slide preparation and counting, since DAPI fluorescence fades less rapidly than acrydine orange. DAPI staining does not allow accurate measurement of the size of the bacterial cells, since it could only stain the specific part of the cell containing DNA [8]. Visualization of bacteria in dense biofilms is highly difficult. This problem can be overcome to a certain extent by using confocal scanning laser microscopy (CSLM) (Chapter 1, part 2). DAPI staining has been intensively used for determination of bacterial abundance in water samples [9] as well as in biofilms [10]. This can be useful for the determination of the efficiency of biocides (Chapter 2).
Figure 1.2 Bacterial cells stained with DAPI visualized under an epifluorescent microscope. Magnification 1000 ×.
Fluorescent in situ hybridization (FISH) allows quick phylogenetic identification (phylogenic staining) of microorganisms in environmental samples without the need to cultivate them or to amplify their genes using the polymerase chain reaction (PCR) [11] (Table 1.2, Figure 1.3). This method is based on the identification of microorganisms using short (15–20 nucleotides) rRNA-complementary fluorescently labeled oligonucleotide probes (species, genes or group specific) that penetrate microbial cells, bind to RNA and emit visible light when illuminated with UV light [12]. Common fluorescent dyes include Cy3, Cy5 and Alexa®. In comparison with other molecular methods (Chapter 3), FISH provides quantitative data about abundance of bacterial groups without PCR bias [13]. The FISH-based protocol is presented later in this chapter (Chapter 1, section 2); here the modified protocol of catalyzed reporter deposition fluorescent in situ hybridization (CARD-FISH) is described. CARD-FISH is based on the deposition of a large number of labeled tyramine molecules by peroxidase activity (Figure 1.3), which enhances visualization of a small, slow growing or starving bacteria that have a small amount of rRNA and, thus, give a weak FISH signal [14]. Additionally, CARD-FISH can be used for the visualization and assessment of the densities of microorganisms in the samples that have high background fluorescence, such as algal surfaces, fluorescent paints, phototrophic biofilms and sediments [14–16]. In this procedure, FISH probes are conjugated with the enzyme (horseradish peroxidase) and after hybridization the subsequent deposition of fluorescently labeled tyramides results in substantially higher signal intensities on target cells [16]. The critical step of CARD-FISH is to ensure probe microbial cell permeability with cellular integrity, especially in diverse, multispecies microbial communities [17]. Recent improvements in CARD-FISH samples preparation, permeabilization and staining techniques have resulted in a significant improvement in detection rates of benthic and planktonic marine bacteria [14, 15].
Table 1.2 Common probes used in FISH and CARD-FISH and their specific conditions. Detailed information about rRNA-targeted oligonucleotide probes can be found in the public database ProbeBase (http://www.microbial-ecology.net/default.asp) [19, 20].
aGAM42a requires competitor GCC TTC CCA CTT CGT TT that increases chances of specific binding.
bBET42a requires competitor GCC TTC CCA CAT CGT TT that increases...
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