IMAGING OF BACTERIAL-CELLS BY FLUORESCENCE EXCLUSION USING SCANNING CONFOCAL LASER MICROSCOPY

Attached bacterial cells, microcolonies and biofilms were studied using negative fluorescent staining (fluorescence exclusion) in conjunction with scanning confocal laser microscopy (SCLM). Use of low molecular weight fluorescent compounds (i.e., fluorescein) provided resolution of the cell boundary suitable for quantitative image analysis of cell size and shape. In contrast with positive fluorescent stains (i.e., acridine orange, fluorescein isothiocyanate), the non-toxic nature of the negative stains also allowed time-courses of microcolony development to be obtained using the SCLM system in conjunction with continuous-flow slide culture. These latter observations indicated that the laser system could be used for temporal studies of microcolony and biofilm development. Low molecular weight fluorophores effectively penetrated thick biofilms (70-mu-m) allowing xy or xz optical sectioning of biofilms at 0.2-mu-m intervals, whereas size fractionated fluorescein isothiocyanate conjugated dextrans exhibited differential penetration. In addition, studies on the effect of fluorophore concentration on image quality indicated a need for optimization to balance image contrast and beam quenching effects.