IN-SITU CLASSIFICATION AND IMAGE CYTOMETRY OF PELAGIC BACTERIA FROM AHIGH-MOUNTAIN LAKE (GOSSENKOLLESEE, AUSTRIA)

We describe a procedure to measure the cell sizes of pelagic bacteria after determinative hybridization with rRNA-targeted fluorescently lab eled oligonucleotide probes. Our approach is based on established imag e analysis techniques modified for objects simultaneously stained with two fluorescent dyes. It allows the estimation of biomass and cell si ze distribution and the morphological characterization of different ba cteriol taxa in plankton samples. The protocol was tested in a study o f the bacterioplankton community of a high mountain lake during and af ter the ice break period. Cells that hybridized with a probe for the d omain Bacteria accounted for 70% of the bacterial abundance (range, 49 to 83%) as determined by 4',6'-diamidino-2-phenylindole staining (K. G. Porter and Y. S. Feig, Limnol. Oceanogr. 25:943-948, 1980), but for >85% of the total biomass (range, 78 to 99%). The size distribution f or members of the beta subclass of the Proteobacteria shifted toward l arger cells and clearly distinguished this group from the total bacter ial assemblage. In the surface water leger beneath the winter cover, b acteria belonging to the beta 1 subgroup constituted about one-half of the beta subclass abundance. The mean cell volume of the beta 1 subgr oup bacteria was significantly less than that of the beta subclass pro teobacteria, and the beta 1 subgroup accounted for less than 30% of th e total beta subclass biovolume. Two weeks later, the biovolume of the beta Proteobacteria had decreased to the level of the beta 1 subgroup , and both the biovolume size distributions and cell morphologies of t he beta Proteobacteria and the beta 1 subgroup were very similar. We c ould thus quantify the disappearance of large, morphologically distinc t beta subclass protcobacteria which were not members of the beta 1 su bgroup during the ice break period. Our results demonstrate that chang es in biovolumes and cell size distributions of different bacterial ta xa, and eventually of individual populations, reveal hitherto unknown processes within aquatic bacterial assemblages and may open new perspe ctives for the study of microbial food webs.