This article was downloaded by:[Callow, Maureen] [Callow, Maureen] On: 12 April 2007 Access Details: [subscription number 776605365] Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Biofouling The Journal of Bioadhesion and Biofilm Research Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713454511 Combinatorial materials research applied to the development of new surface coatings V. Application of a spinning water-jet for the semi-high throughput assessment of the attachment strength of marine fouling algae To cite this Article: , 'Combinatorial materials research applied to the development of new surface coatings V. Application of a spinning water-jet for the semi-high throughput assessment of the attachment strength of marine fouling algae', Biofouling, 23:2, 121 - 130 To link to this article: DOI: 10.1080/08927010701189583 URL: http://dx.doi.org/10.1080/08927010701189583 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article maybe used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. © Taylor and Francis 2007 Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 Biofouling, 2007; 23(2): 121 – 130 Combinatorial materials research applied to the development of new surface coatings V. Application of a spinning water-jet for the semi-high throughput assessment of the attachment strength of marine fouling algae FRANCK CASSÉ1, SHANE J. STAFSLIEN2, JAMES A. BAHR2, JUSTIN DANIELS2, JOHN A. FINLAY1, JAMES A. CALLOW1 & MAUREEN E. CALLOW1 1 The University of Birmingham, School of Biosciences, Birmingham, UK, and 2Center for Nanoscale Science and Engineering, North Dakota Sate University, Fargo, North Dakota, USA (Received 4 October 2006; accepted 21 December 2006) Abstract In order to facilitate a semi-high throughput approach to the evaluation of novel fouling-release coatings, a ‘spinjet’ apparatus has been constructed. The apparatus delivers a jet of water of controlled, variable pressure into the wells of 24-well plates in order to facilitate measurement of the strength of adhesion of algae growing on the base of the wells. Two algae, namely, sporelings (young plants) of the green macroalga Ulva and a diatom (Navicula), were selected as test organisms because of their opposing responses to silicone fouling-release coatings. The percentage removal of algal biofilm was positively correlated with the impact pressure for both organisms growing on all the coating types. Ulva sporelings were removed from silicone elastomers at low impact pressures in contrast to Navicula cells which were strongly attached to this type of coating. The data obtained for the 24-well plates correlated with those obtained for the same coatings applied to microscope slides. The data show that the 24-well plate format is suitable for semi-high throughput screening of the adhesion strength of algae. Keywords: Alga, diatom, fouling release, high-throughput screen, silicone elastomer, Ulva, Navicula Introduction All surfaces placed in the sea are rapidly colonised by a consortium of marine organisms specialised for benthic life. Ships and other marine structures are traditionally protected from biofouling by biocidecontaining antifouling paints (Turley et al. 2005; Finnie, 2006, Jelic-Mrcelic et al. 2006) but new coatings are now required that do not have a negative impact on the marine environment. The only major type of non-biocidal coatings currently commercially available are based on elastomeric polydimethylsiloxane (PDMS), the so-called fouling release coatings (e.g. Kavanagh et al. 2001, Stein et al. 2003, Sun et al. 2004, Wendt et al. 2006). Fouling release coatings facilitate the weak adhesion of macrofouling organisms such as barnacles, tubeworms and macroalgae (Holm et al. 2006), which are released under suitable hydrodynamic conditions (Kavanagh et al. 2005). Finding alternative tech- nologies is expensive and time consuming as the number of combinations that need to be synthesised, characterised and evaluated is vast. Evaluation of coatings applied to microscope slides has been used successfully to reveal the strength of attachment of algal biofilms when a limited number of coatings are being studied (e.g. Chaudhury et al. 2005; Gudipati et al. 2005; Tang et al. 2005; Krishnan et al. 2006a; 2006b; Statz et al. 2006; Yarbrough et al. 2006). However, the combinatorial approach adopted by North Dakota State University (NDSU) has the potential to generate hundreds of combinations of polymers of the same generic type (Webster et al. 2004; Webster 2005; 2007). Hence, new screening methods are required to down-select samples with foulingrelease potential to a number that is manageable for more extensive biological evaluation, e.g. through more rigorous assays of coatings applied to slides or raft panels. Correspondence: M. E. Callow, The University of Birmingham, School of Biosciences, Birmingham B15 2TT, UK. Fax: þ44(0) 121 414-5447. E-mail: m.e.callow@bham.ac.uk ISSN 0892-7014 print/ISSN 1029-2454 online Ó 2007 Taylor & Francis DOI: 10.1080/08927010701189583 Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 122 F. Cassé et al. Large scale screening of bioactive compounds using high-throughput methods are now routinely used in the pharmaceutical industry. Such methods using multi-well plates and plate readers have also been adapted successfully for screening bioactive compounds against fouling organisms (Bers et al. 2006; Stafslien et al. 2006; 2007a). The present paper describes a semi-high throughput method based on 24-well plates to determine the strength of attachment of algae. The data are compared to those obtained for the same coatings applied to glass microscope slides. Two types of fouling algae with different adhesion characteristics were selected, namely, the green macroalga Ulva linza and a diatom, Navicula perminuta. Ulva (syn. Enteromorpha) is the most common macroalga that fouls ships and other submerged structures. Dispersal of Ulva is mainly through motile, quadriflagellate zoospores (approximately 7 – 8 mm in length), which are released in large numbers and form the starting point of the assay (Callow et al. 1997). The swimming spores settle and adhere to suitable surfaces, adhesion being mediated through a glycoprotein adhesive (Callow & Callow, 2006). The settled spores rapidly germinate into sporelings (young plants), which adhere weakly to fouling release coatings (Schultz et al. 2003; Chaudhury et al. 2005). Slimes dominated by diatoms are the predominant form of microfouling on all illuminated surfaces immersed in the sea (Patil & Anil 2005a; 2005b), including biocidal antifouling paints (Cassé & Swain, 2006; Jelic-Mrcelic et al. 2006) and non-biocidal coatings (Terlizzi et al. 2000; Cassé & Swain, 2006). Adhesion is especially tenacious to silicone fouling-release coatings and diatom slimes are not released from vessels including those that operate at high speeds (Terlizzi et al. 2000; Holland et al. 2004). The ease of removal of biomass from surfaces was quantified by application of hydrodynamic forces using a miniaturised water-jet apparatus (‘spinjet’), specially designed for use on 24-well plates. Methods Sample preparation A number of calibration experiments were performed using untreated 24-well plates (3524, Corning Incorporated, Costar1). The details of individual experiments are provided in the Results section. The coatings used in the assays comprised two foulingrelease siloxanes, namely, Dow Corning’s Silastic1 T-2 and Intersleek1 (International Paint Ltd) and polyurethane. The coatings were either applied to glass or aluminium discs that were fixed in the wells or were directly deposited in the wells as described by Stafslien et al. (2006). Standard coatings were applied to glass slides and 24-well plates at NDSU. Prior to coating preparation, glass slides were immersed for 24 h in a 1:3 solution of hydrogen peroxide and sulphuric acid, respectively (piranha solution). Slides were removed from the piranha solution and immediately rinsed with copious amounts of deionised water and dried at ambient laboratory conditions. Coatings solutions were then dispensed on piranha treated slides until complete coverage was achieved. Coatings were applied to 15 mm aluminium discs already fixed onto the bottom of the wells with epoxy for the 24-well plates as described in Stafslien et al. (2006). In some experiments, coatings were applied to 15 mm diameter glass coverslips that were subsequently adhered to the bottom of the wells using colourless, white or black epoxy. Preliminary experiments showed no significant difference between results obtained for both formats. Leaching of test samples All 24-well plates were vented for 1 week in a flow oven at 308C and then pre-leached in deionised water for 4 weeks with daily exchange of water in the wells at NDSU prior to shipping to Birmingham (Stafslien et al. 2006). The slides with experimental coatings were shipped to Birmingham where they were leached for 4 weeks in a 30-l tank of recirculating deionised water fitted with a carbon filter. The 24-well plates and slides were equilibrated in artificial seawater for 2 h before the start of each experiment. Ulva sporeling assay in 24-well plates Fertile Ulva linza was collected from Wembury Beach, England (508180 N; 48020 W) and zoospores were released as described in Callow et al. (1997). The concentration of spores was adjusted to 56105 spores ml71. Twelve replicates wells (6 per row) were used for each treatment. Each well of the 24-well plates was inoculated with 1 ml of zoospore suspension. The 24-well plates were immediately placed for 2 h in the dark at 208C to allow the spores to settle. The wells were then emptied to remove unsettled spores and 1 ml of enriched seawater medium (Starr & Zeikus, 1987) was added per well. The plates were placed in an illuminated incubator at 188C with a 16:8 light: dark cycle (photon flux density 46 mmol m72 s71) for 5 d and the medium changed every 48 h. The strength of attachment of the biomass was determined using the spinjet apparatus described below. The 24-well plates were jetted at a range of Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 Assessment of attachment strength using a spinning water-jet 123 impact pressure. One row of 6 replicates was jetted; the adjacent row was not jetted. Non-jetted samples provide information on the amount of biomass in the wells and also serve as controls for calculation of percentage biomass removal. Biomass was quantified by the fluorescence of chlorophyll, which was extracted from biomass in the well with 1 ml of dimethyl sulphoxide (DMSO). The plates were incubated in darkness for 30 min. After ensuring adequate mixing, 200 ml of DMSO were pipetted from each well into wells of a 96-well plate and the fluorescence read in a Tecan plate reader (GENios Plus) with chlorophyll filter (excitation wavelength: 360 nm; emission wavelength: 670 nm) connected to a computer with Magellan v.4.00 software. Each well reading was based on four spot readings, taken in a 262 square. All plates were read from the top. Fluorescence was recorded as Relative Fluorescence Units (RFU). The mean of six replicate wells + 95% confidence limits was calculated. The strength of attachment data are presented as percentage removal compared with the controls, +95% confidence limits derived from arcsine transformed data. The distribution of spores deposited in the uncoated polystyrene wells after 2 h settlement in darkness was quantified on untreated plates. After washing, the attached spores were fixed in 2.5% glutaraldehyde in seawater (Callow et al. 1997). Settled spores, viewed through the bottom of the wells, were counted at 1 mm intervals across the diameter of the well as described in Callow et al. (2002). described by Finlay et al. (2002), which was adapted for use with microscope slides from the original apparatus (Swain & Schultz, 1996). RFU readings (80 per slide) were taken from the central part of the slide that was exposed to the water jet. The percentage removal was calculated as described above. Ulva sporeling assay on coatings applied to microscope slides Diatom assays on coatings applied to microscope slides Coated slides (6 replicates per treatment) were placed in individual compartments of Quadriperm dishes (Greiner) and 10 ml of a spore suspension containing 56105 spores ml71 added. After 3 h in darkness, the slides were washed in artificial seawater (ASW) to remove any unattached spores. The settled spores were cultured (Chaudhury et al. 2005) for 5 days under the same conditions as the 24-well plates. Growth was estimated by direct measurement of fluorescence from the chlorophyll of the sporelings using a Tecan plate reader (GENios Plus). Fluorescence was recorded as RFU from direct readings. The slides (6 replicates) were read from the top, 300 readings per slide, taken in blocks of 30610. One blank slide of the same coating was used to obtain a mean background reading and this value was subtracted from the respective test surfaces. The strength of attachment of the sporelings was determined by jet washing using the water jet Diatom assays in 24-well plates The diatom Navicula perminuta was cultured in natural seawater supplemented with nutrients from Guillard’s F/2 medium as described in Holland et al. (2004). Cultures were grown under static conditions in 250 ml Pyrex conical flasks containing 100 ml medium in a growth cabinet at 188C with a 16:8 light: dark cycle (photon flux density 21 mmol m72 s71). The cell suspension was poured away leaving a biofilm of cells adhered to the bottom of the flasks. The biofilm was gently resuspended in artificial seawater (ASW) before filtering through 20 mm nylon mesh. The concentration of cells was adjusted to 46105 cells ml71. Each 24-well plate was inoculated with 1 ml of cell culture, which was left for 2 h on the laboratory bench in the light at room temperature. Quantification of biomass and the adhesion assay were the same as described above for Ulva. The distribution of Navicula cells in uncoated polystyrene wells after spinjetting was determined after fixing in 2.5% glutaraldehyde in seawater as described for Ulva. Six replicate slides of each surface placed into individual compartments of Quadriperm dishes (Greiner) were inoculated with 10 ml of culture containing 46105 cells ml71. The dishes were allowed to stand for 2 h on the bench in the light at room temperature. Strength of attachment of cells was determined by jet washing 3 replicate slides with artificial seawater using the water jet described by Finlay et al. (2002). The other 3 slides served as controls. Cells were fixed in 2.5% glutaraldehyde in seawater, rinsed in deionised water and air dried. The number of cells attached was counted using a Zeiss Kontron 3000 image capture analysis system attached to a Zeiss epifluorescence microscope (Callow et al. 2002). Counts were made for 30 fields of view within the area of the slide that was exposed to the water jet. The number of cells was compared to counts made on the three unexposed samples. The percentage removal was calculated from the mean of cell density before and after jet washing. Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 124 F. Cassé et al. Spinjet apparatus The apparatus was designed to evaluate the strength of attachment of microorganisms in 24-well plates using a perpendicular water jet that impacts the bottom of the coated well. The Spinjet represents further development and miniaturisation of previously described water jets designed for use with microscope slides (Finlay et al. 2002) and raft panels (Swain & Schultz, 1996). The jet of water was produced by a straight nozzle placed eccentrically on a rotating shaft, thus the nozzle rotation describes a circle of 7 mm in diameter inside of a 15 mm diameter well (Figure 1). Well plates were loaded manually into the indexing plate of the Spinjet and clamped in place. The gas supply pressure was then adjusted to the desired jetting pressure with the precision pressure regulator. An integral precision test gauge allowed the pressure to be set to within +3.5 kPa out of a total range of 0 – 1034 kPa. The jet duration was then entered in seconds into the digital timing relay to an accuracy of 100 ms. Each well jetting was then triggered with a pneumatic foot switch tethered to the timing relay. The relay also controlled the starting and stopping of the nozzle rotation (Figure 2). Calibration of the Spinjet nozzle was performed against an analytical balance to generate the relation- ship between the set dispense pressure and flow rate over time. Single volumes of jetted water were collected at specific pressures and jet durations and weighed with the analytical balance. The flow rates calculated from these measured volumes were then divided by the flow area of the nozzle to calculate the average velocity of the water jet exiting the nozzle. The average water jet velocities were then used to calculate the impact pressures impinging on the well bottoms (Finlay et al. 2002) where r is the fluid density. Impact pressure ¼ 1=2 r ðAverage jet velocityÞ2 For the given geometries, a supply pressure of 100 kPa produced an impact pressure of 67 kPa (Figure 3). The plates were placed into the water jet holder and treated at a range of impact pressures with artificial seawater (Instant Ocean) for 10 s per well. Time course experiments had established that increasing the duration of jetting beyond 10 s did not influence the percentage removal, maximum cell removal being obtained after 10 s at any single pressure. For each treatment or experimental coating, 12 replicates were used; one line of six replicate wells was sprayed per impact pressure and the adjacent six served as unsprayed controls. Figure 1. Spinjet description. (a) Spinjet overview; (b) uncoated 24-well plate loaded into the Spinjet with the water jet on; (c) representation of the Spinjet off-set nozzle and well geometries. The plates are inverted on the platform and the Spinjet sprays the water into the well from below via an offset nozzle. Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 Assessment of attachment strength using a spinning water-jet 125 Figure 2. Spinjet process and instrument diagram. Dispensing pressure is supplied from a compressed gas connection to the precision pressure regulator. Water jetting pressure is then manually set with the precision pressure regulator. Jet duration and rotation are controlled by the digital timing relay, triggered by a foot switch, while the nozzle rotates at 120 rpm. Figure 3. Typical calibration data for the Spinjet. Resultant impact pressures generated from various supply pressures (dispense tank pressure). Test repeated 3 times on same nozzle with a maximum SD of 1.02. Figure 4. Mean number of attached spores per mm2 after 2 h settlement in the dark. Counts were taken at 1 mm intervals from the midpoint of the well (zero). Each point is the mean count from 6 replicate wells. Bars ¼ +95% confidence limits. Results Distribution of settled Ulva spores The mean spore count taken from transects across the diameter of wells is presented in Figure 4. The plot shows that spores were evenly distributed across the well and had not settled preferentially at the sides of the well. Effect of background colour on growth and attachment strength of Ulva sporelings Ulva sporelings were cultured on Silastic1 T2 and polyurethane applied to glass coverslips which were secured to the bottom of wells by either white or black epoxy. Biomass data after 5 days of growth on white vs. black backgrounds is shown in Table I. Although the total amount of biomass was less on the Silastic1 T-2 than the polyurethane, there was no significant difference between the amount of biomass developed in relation to background colour. The percentage removal of biomass increased with increasing impact pressure on both Silastic1 T2 and polyurethane (Figure 5). Biomass was removed from the fouling release coating (Silastic1T-2) at a lower impact pressure than from the polyurethane coating (Figure 6). At the highest impact pressure tested (152 kPa) only 40% of the biomass was removed from the polyurethane compared to over 80% from Silastic1 T-2. The strength of attachment of the biomass was not significantly different on black vs. white surfaces (Table I). Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 126 F. Cassé et al. Table I. Biomass of Ulva after 5 d growth (RFU) and percentage removal at 75 kPa of impact pressure on white vs black backgrounds. RFU means are from 6 replicate wells +95% confidence limits. Percentage removal data are based on 6 unjetted replicates and 6 spinjetted replicates. Paired t-tests show no significant difference between growth or percentage removal on black vs. white surfaces for either polyurethane or Silastic1 T-2. Biomass White + 95% conf. limits Biomass Black + 95% conf. limits % removal White + 95% conf. limits % removal Black + 95% conf. limits Polyurethane Silastic1 T2 34805 + 1117 28193 + 2978 35345 + 885 29192 + 2287 21.7 + 4.2 65.7 + 4.1 19.7 + 5.0 64.1 + 5.8 Attachment strength of Ulva sporelings to coatings on 24-well plates and slides The percentage removal of Ulva sporelings growing on glass and two silicone elastomers, Silastic1 T2 and Intersleek1 deposited in 24-well plates and on coated microscope slides is shown in Figure 7. The impact pressure used for the 24-well plates generated by the spinjet was 89 kPa and slides were subjected to 73 kPa impact pressure from a water jet. These data are representative of those obtained in three separate experiments. Figure 7 shows that the coatings rank in the same order in terms of biomass removal and a similar percentage removal of biomass was obtained by both methods of coating application. However, since the impact pressures used are slightly different for the spinjet and the water jet, it is not appropriate to directly compare the two data sets. Adhesion strength of Navicula and distribution of cells after spinjetting Figure 5. Percentage removal of Ulva sporeling after 5 d growth on coatings deposited in 24-well plates and hosed at 18, 43, 75, 111 and 152 kPa impact pressure with the Spinjet. Each point is the mean of 6 replicate wells. Bars ¼ +95% confidence limits derived from arcsine transformed data. Adhesion strength, expressed as percentage removal, is presented in Figure 8. These data are representative of those obtained in three separate experiments. Approximately 80% of the cells were removed from polyurethane by an impact pressure of 18 kPa compared to 30% from Silastic1 T-2. Cell counts across the diameter of uncoated polystyrene wells sprayed at three pressures are presented in Figure 9. The density of cells remaining is fairly uniform; at the two lowest pressures, namely, 18 and 43 kPa, 27% and 16%, respectively, of the original biomass remained. At the highest pressure (152 kPa), the cell density was lower around Figure 6. Photograph of a 24-well plate after 5 d growth of Ulva sporeling (row 1 and 3) and after jetting at 42 kPa impact pressure with the Spinjet (row 2 and 4). Wells in the top two rows contained glass coverslips coated with polyurethane and the bottom two rows contained coverslips coated with Silastic1 T2. The glass coverslips were secured in the wells with clear epoxy glue; 20% and 60% of the biomass were removed by jetting from the polyurethane and Silastic-T2, respectively. Figure 7. Percentage removal of Ulva sporeling after 5 d growth on coatings deposited in 24-well plates or on glass microscope slides. The two experimental coatings were deposited directly onto aluminium discs that were secured in the 24-well plates with epoxy glue. The wells were subjected to an impact pressure of 89 kPa with the Spinjet and the slides to 73 kPa with the water jet. Each point is the mean of 6 replicates. Bars ¼ +95% confidence limits derived from arcsine transformed data. Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 Assessment of attachment strength using a spinning water-jet Figure 8. Percentage removal of Navicula after 2 h settlement on coatings deposited in 24-well plates and jetted at 18, 43, 67, 89 and 111 kPa impact pressure with the Spinjet. Each point is the mean of 6 replicate wells. Bars ¼ +95% confidence limits derived from arcsine transformed data. 127 Figure 10. Percentage removal of Navicula after 2 h settlement on coatings deposited in 24-well plates or on glass microscope slides. Aluminium discs were secured in the 24-well plates with epoxy glue. The wells were subjected to an impact pressure of 31 kPa with the Spinjet and the slides to 34 kPa with the water jet. Each point is the mean of 6 replicate wells for the plates and 90 counts on 3 replicates for the slides. Bars ¼ +95% confidence limits derived from arcsine transformed data. plates (F (2, 15) ¼ 160, p 5 0.05) and slides (F (2, 267) ¼ 641, p 5 0.05). Discussion Figure 9. Navicula cell density obtained from cell counts across the middle of polystyrene wells subjected to different impact pressures; 18 kPa, 43 kPa and 152 kPa impact pressures show respectively 73%, 84% and 94% removal of cell biomass based on chlorophyll extraction. Counts were taken at 1 mm intervals. Each point is the mean of 6 replicate wells. Bars ¼ +95% confidence limits. the zone of impact of the water jet; 94% of the cells were removed at this impact pressure. Attachment strength of Navicula to coatings on 24-well plates and slides The percentage removal of Navicula from glass and two silicone elastomers, Silastic1 T-2 and Intersleek1 deposited in 24-well plates and applied to microscope slides is shown in Figure 10. The impact pressure used for the 24-well plates generated by the spinjet was 31 kPa and slides were subjected to 34 kPa impact pressure from a water jet. The rank order in terms of percentage removal from the coatings was the same for both methods of coating application although the percentage of biomass removed was slightly higher for the two silicone elastomers in 24-well plates compared to the slides. There was a significant difference between glass, Silastic1 T-2 and Intersleek1 for both the 24-well Algal biofilms develop on all artificial surfaces immersed in the sea provided there is some illumination (Callow, 2000). Adhesion of diatoms is moderated by the production of extracellular polymeric substances (EPS) comprising various polysaccharide and glycoprotein components (Chiovitti et al. 2006). Macroalgal fouling communities are frequently dominated by Ulva, which has a different type of adhesion biology to that of diatoms (see Callow & Callow, 2006). The difference in the adhesion biology of these two types of fouling algae necessitates the development of bioassays suitable for both organisms, since Ulva sporelings adhere only weakly to silicone elastomers (Chaudhury et al. 2005) while diatoms adhere relatively strongly to these coatings (Holland et al. 2004). The development of coatings to which the adhesion of both macro- and microfouling organisms is weak is a target for coatings development (see Krishnan et al. 2006a; 2006b). A fouling release assay employing Ulva sporelings and diatoms in 24-well plates provided a convenient and reproducible semi-high throughput method for screening coatings in terms of their strength of attachment. The 24-well plate format allowed good replication and the data have been shown to be reproducible. Moreover the large number of individual test samples that can be assayed simultaneously, under the same conditions, increases the usefulness of the method. Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 128 F. Cassé et al. Preliminary data showed that for the method to be used reliably, venting and leaching of the 24-well plates must be considered carefully. One week of venting in an air flow cabinet followed by leaching for 4 weeks with daily water exchange appeared to be adequate for most of the surfaces, including silicone elastomers cured with dibutyltin compounds. Ulva spores settled evenly on the surface of the wells provided a low spore inoculum was employed. Spores are known to respond to topographic features (Hoipenkeimer-Wilson et al. 2004; Carman et al. 2006) and at high concentrations (416106 ml71), they settle preferentially around the edges of the wells. Germination and growth were not significantly affected by background colour in the 24-well plates, in contrast to slides, where a significantly slower rate of spore germination and growth on black surfaces compared to white surfaces are seen (unpublished data). Since the strength of adhesion of Ulva sporelings to fouling release silicone elastomers is also related to the stage of growth (Schultz et al. 2003), the 24-well plate format would be preferred to a slide format if dark-coloured coatings were being investigated. The different performance of the two methods in relation to background colour may be due to the different reflective properties of the multiwell plates compared with glass slides, the former having many reflective surfaces that may serve to minimise differences in light fields experienced by the organisms. The influence of background colour on the development of algal fouling on panels immersed in the ocean has recently been shown by Swain et al. (2006). Adhesion strength, measured as percentage removal of biomass, was positively correlated with impact pressure provided by the spinjet for both algae. Moreover, the percentage removal data for the 24-well plates strongly correlated with those obtained for the same coatings applied to glass microscope slides, which were hosed using a standard water jet (Finlay et al. 2002). Ulva sporelings were weakly attached to the silicone elastomers (Silastic1 T-2 and Intersleek) and strongly attached to glass, which concurs with previous observations (Chaudhury et al. 2005; Krishnan et al. 2006a; 2006b). Conversely, diatoms adhered relatively strongly to the silicone elastomers compared to glass, in agreement with published data (Holland et al. 2004). All of the adhesion strength data from 24-well plates concurs with those obtained previously for Ulva (Chaudhury et al. 2005; Krishnan et al. 2006a) and diatoms (Holland et al. 2004). Cell counts across the bottom of wells containing adhered diatoms following exposure to different spinjet pressures showed that cell removal was broadly even across the diameter of the well. The differential removal seen at the highest impact pressure is not considered to be important since down-selection of formulations would never be based on percentage removal values 490% in view of the asymptotic nature of the removal curves. Visual observation of Ulva biofilms also indicated an even removal of biofilm from the well. Shear forces produced by the impinging jet on the biofilm samples are concentrated in a circular region of high shear that reaches a maximum value at: tmax ¼ 0:32ðimpact pressure of jetÞ=ðH=dÞ2 where H ¼ distance from nozzle to surface, d ¼ nozzle diameter and impact pressure is 1/2 rV2 (Beltaos & Rajaratnam, 1974). Although this equation is for a static non-rotating jet, it can be applied to the Spinjet due to the relatively slow rotational speed of the nozzle. For example, the rotational speed of the impact region of the water jet is 4.4 cm s71 as it traces a 7 mm diameter circle in the well bottom and the velocity of the water jet is almost three orders of magnitude higher than the rotational velocity at 1000 – 3000 cm s71 over the pressure range used for testing. Therefore, it is assumed that the rotation speed of the jet has a negligible addition to the speed of the impacting water jet at the leading edge of rotation and the radial shear region is approximately symmetrical. With the given nozzle geometries an impact pressure of 50 kPa will produce a tmax of *15 Pa. The circular high shear region is *3 – 4 mm in diameter and sweeps out an area equal to *55% of the total well bottom area. The remainder of the well bottom is cleaned less vigorously by shear forces that are less than tmax. The nozzle rotates within the well twice a second or 20 times during a typical 10 sec jetting to ensure that the wells are exposed to a repeatable shearing action regardless of nozzle starting and end position. The complete mechanism of biofilm removal from an impinging jet involves the shear force as well as the normal force of the jet as it impinges on the coating sample. The resultant removal force on the biofilm is quite complex and not fully understood. Modelling of the water jet shear forces and how they correlate to those experienced on the side of a ship was outside the scope of this work. However, the goal of the Spinjet design was to deliver a consistent water jet shearing action (sum of all forces) at a specified supply pressure so that coatings could be ranked by their relative cleanability. This was accomplished through the close control of water jet pressure, water jet duration, and water jet impingement region reproducibility. In summary, the data have shown that the spinjet delivers a suitable range of impact pressures that facilitate measurement of the adhesion strength of algae to non-biocidal coatings deposited in 24-well Downloaded By: [Callow, Maureen] At: 13:01 12 April 2007 Assessment of attachment strength using a spinning water-jet plates. The plate format is also suitable to monitor the efficacy of fouling release coatings that incorporate a tethered biocide (Thomas et al. 2004). The combination of 24-well plate assays employing bacteria (Stafslien et al. 2007a; 2007b) and algae will contribute data on which the down-selection of coatings for further development can be based. Acknowledgements This study was carried out with support from the US Office of Naval Research in the form of grants N00014-03-1-0509 to JAC & MEC and N00014-021-0794 and N00014-03-1-0702 to NDSU. References Beltaos S, Rajaratnam N. 1974. Impinging circular tubular jets. ASCE J Hydrauic Div 100:1313 – 1328. Callow ME. 2000. Algal biofilms. In: Evans LV, editor. Biofilms: recent advances in their study and control. Amsterdam: Harwood Academic Publishers. pp 189 – 209. Bers AV, D’Souza F, Klijnstra JW, Willemsen PR, Wahl M. 2006. Chemical defence in mussels: antifouling effect of crude extracts of the periostracum of the blue mussel Mytilis edulis. Biofouling 22:251 – 260. Callow JA, Callow ME. 2006. The Ulva spore adhesive system. In: Smith AM, Callow JA, editors. Biological adhesives. 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