title: Bacterial assay for the rapid assessment of antifouling and fouling release properties of coatings and materials.
abstract: 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.
authors.affiliation: Corrosion and Antifouling, TNO Science and Industry, Bevesierweg MML (Harssens), Den Helder, The Netherlands. fraddry.dsouza@tno.nl
authors.forename: Fraddry
authors.initials: F
authors.lastname: D'Souza
authors.forename: Anouk
authors.initials: A
authors.lastname: Bruin
authors.forename: Rens
authors.initials: R
authors.lastname: Biersteker
authors.forename: Glen
authors.initials: G
authors.lastname: Donnelly
authors.forename: Job
authors.initials: J
authors.lastname: Klijnstra
authors.forename: Corne
authors.initials: C
authors.lastname: Rentrop
authors.forename: Peter
authors.initials: P
authors.lastname: Willemsen
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development*; D015169:Colony Count, Microbial / Q000379:methods; D004783:Environmental Microbiology; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000494:pharmacology; D044129:Halomonadaceae / Q000187:drug effects* / Q000254:growth & development* / Q000378:metabolism; D009930:Organic Chemicals / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C461159:SYTO 13