Use of confocal laser scanning microscopy for biofilm investigation on paints under field conditions

Abstract To investigate biofilm growth on paints under field conditions, we developed a modified approach enabling subsequent Confocal Laser Scanning Microscopy (CLSM) for the three-dimensional analysis of undisturbed samples. Painted polycarbonate slides were glued on polycarbonate panels mounted on a test raft in a harbour, and one slide per paint and depth was used on each of the five sampling dates over three months. On seven out of ten paints, biomass dry weight and chlorophyll a of biofilms increased nearly linearly over time whereas biofilm development was inhibited on three paints. Quantitative CLSM results revealed that extracellular polymeric substances were the dominating component of the biofilms. Since top-view CLSM did not enable the complete observation of thick biofilms, cryosections were prepared to determine biofilm thickness. Results of biofilm thickness were comparable to those of biomass and chlorophyll. The vertical extension of CLSM top-view images was not related to biomass dry weight and to biofilm thickness from cryosections and was, therefore, no suitable measure for biofilm characterisation. In contrast, biofilm thickness measured from cryosections was positively related to biomass dry weight indicating the need of cryosections for thick biofilms.