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

Confocal laser scanning rnicroscopy (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 llght and phase contrast microscopy and scanning electron microscopy. Confocal microscopy proved superior in sevcrdl respects: ( l ) It gave clear images of organisms even if they were overlaid with a thick layer of mucus, other organlsms and debr~s. (2 ) It did not require the biofilm to be disrupted or dned 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 biofdm to be v~sualised. 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 a n invaluable aid for examining the structure and growth of living biofilrns in studies of shore ecology and marine fouling. varying seasonally and in different habitats (Underwood 1984, Hill & Hawkins 1991, Anderson 1995). The organisms may be very abundant; estimates of 3 X 107 bacteria (Hendy 1951) and 2.7 x 105 diatoms cm-2 (Edyvean & Moss 1986) have been made for marine