The in vitro settlement assay using cyprids of Balanus. amphitrite is an important tool in basic and applied research. The aim of this study was to quantify the effect of gregariousness within these assays, and to determine the interaction between gregariousness and container size, and settlement promoters and inhibitors. Assays with 1, 5, 10, 15, 20, and 40 cyprids in containers with a log range of surface area to volume ratios were conducted in a fully factorial design. Assays with the same range of cyprid numbers and six concentrations of the settlement promoter 3-isobutyl-1-methylxanthine (IBMX) or the inhibitor phloroglucinol were also conducted in a fully factorial design. Percentage settlement was analysed by GLM ANOVA. Significant (p < 0.05) gregarious effects were detected at > or = 5 cyprids in a well. Surface area:volume ratio had a strong effect on cyprid settlement, but this effect could be masked by overcrowding in very small wells. Gregarious interactions between only five cyprids magnified the effect of IBMX by a factor of 10, whereas phloroglucinol had no effect without gregarious interactions. The cyprid settlement bioassay is a valuable tool for basic and applied research but must be used with care.

An assay has been developed to accurately quantify the growth and release behaviour of bacterial biofilms on several test reference materials and coatings, using the marine bacterium Cobetia marina as a model organism. The assay can be used to investigate the inhibition of bacterial growth and release properties of many surfaces when compared to a reference. The method is based upon the staining of attached bacterial cells with the nucleic acid-binding, green fluorescent SYTO 13 stain. A strong linear correlation exists between the fluorescence of the bacterial suspension measured (RFU) using a plate reader and the total bacterial count measured with epifluorescence microscopy. This relationship allows the fluorescent technique to be used for the quantification of bacterial cells attached to surfaces. As the bacteria proliferate on the surface over a period of time, the relative fluorescence unit (RFU) measured using the plate reader also shows an increase with time. This was observed on all three test surfaces (glass, Epikote and Silastic T2) over a period of 4 h of bacterial growth, followed by a release assay, which was carried out by the application of hydrodynamic shear forces using a custom-made rotary device. Different fixed rotor speeds were tested, and based on the release analysis, 12 knots was used to provide standard shear force. The assay developed was then applied for assessing three different antifouling coatings of different surface roughness. The novel assay allows the rapid and sensitive enumeration of attached bacteria directly on the coated surface. This is the first plate reader assay technique that allows estimation of irreversibly attached bacterial cells directly on the coated surface without their removal from the surface or extraction of a stain into solution.