Using confocal laser scanning microscopy, scanning electron microscopy and phase contrast light microscopy to examine marine biofilms

Confocal laser scanning microscopy (CLSM) was used to examine living organisms within marine biofilms growing on microscope slides and on natural opaque and uneven substrata such as rocks and shells. The results were compared to images of identical fields observed under transmitted light and phase contrast microscopy and scanning electron microscopy. Confocal microscopy proved superior in several respects: (1) It gave clear images of organisms even if they were overlaid with a thick layer of mucus, other organisms and debris. (2) It did not require the biofilm to be disrupted or dried and allowed samples to be re-examined at intervals. (3) It could also distinguish living (fluorescing) organisms from dead cells or inorganic matter. (4) Examining optical 'slices' of samples allowed the 3 dimensional structure of the biofilm to be visualised. However, species identification, particularly of diatoms, was much easier using scanning electron microscopy. Methods were developed for overlaying a fixed grid on samples so that specific sites or individual organisms could be accurately relocated for re-examination. Appropriate staining methods were also evaluated. Confocal microscopy will prove to be an invaluable aid for examining the structure and growth of living biofilms in studies of shore ecology and marine fouling.