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

Confocal laser scanning microscopy (CLSM) was used to examine living organi sms within marine biofilms growing on microscope slides and on natural opaq ue and uneven substrata such as rocks and shells. The results were compared to images of identical fields observed under transmitted light and phase c ontrast microscopy and scanning electron microscopy, Confocal microscopy pr oved superior in several respects: (1) It gave clear images of organisms ev en if they were overlaid with a thick layer of mucus, other organisms and d ebris. (2) It did not require the biofilm to be disrupted or dried and allo wed samples to be re-examined at intervals. (3) It could also distinguish l iving (fluorescing) organisms from dead cells or inorganic matter. (4) Exam ining optical 'slices' of samples allowed the 3 dimensional structure of th e biofiIm to be visualised. However, species identification, particularly o f 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. App ropriate staining methods were also evaluated. Confocal microscopy will pro ve to be an invaluable aid for examining the structure and growth of living biofilms in studies of shore ecology and marine fouling.