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.

Anti-Biofilm Performance of Three Natural Products against Initial Bacterial Attachment

“…Both species have previously been used as models in studies on initial attachment of marine bacteria to surfaces [75–77]. Marine bacteria species are commonly cultured in marine broth, which includes peptone (P) as a carbon source recommended for ATCC species, in either sterilised natural sea water (NSW) or artificial sea water (ASW).…”

Life under flow: A novel microfluidic device for the assessment of anti-biofilm technologies

“…For these proof-of-concept experiments, a single flow rate was applied; however, the device is designed to allow flow rates as high as 15 ml min À1 that will result in shear stresses of up to 427 Pa (20.27 knots). The reservoir contained freshly prepared media, i.e., Sea Salts (S9883, Sigma Aldrich) plus 18 g l À1 peptone (SeaSalts Peptone, SSP), 32 and aeration was achieved through a sterile tube entering the reservoir allowing air exchange with the environment. A 0.22 lm sterile filter was adjusted at the end of the tubing to prevent contamination from the surrounding environment during the aeration process.…”

Microfluidic Assay to Quantify the Adhesion of Marine Bacteria

“…Most assays described in literature prefer to work with bacteria in the log phase for adhesion experiments [40] as the results are most reliable. To bring bacteria into the log phase for our microfluidics experiments, the overnight culture was diluted 1:100 in MB and held in liquid culture for approximately 3 h. After this, the OD was frequently measured until the desired OD600 of 0.1 was reached.…”