New image analysis tool to study biomass and morphotypes of three major bacterioplankton groups in an alpine lake

We present an image analysis routine to de- termine the contribution of distinct morphotypes to the total abundance and biomass (carbon) of freshwater bac- terioplankton and to the fraction of cells detected by fluorescence in situ hybridization via catalyzed reporter deposition (CARD-FISH). The method was tested on bacterial assemblages from an alpine lake (Piburger See, Austria) at characteristic time points during the lim- nological year. Although on average 51% of 4',6'-di- amidino-2-phenylindole (DAPI)-stained objects were hy- bridized with the oligonucleotide probe EUB I-III, we detected on average 80% of total biomass determined from DAPI staining. The assemblage was numerically dominated by cocci and rods <0.6 µm (mean cell volume = 0.024 µm 3 ). Only a minor part of these morphotypes could be hybridized (18 and 50%, respectively). In con- trast, larger rods (0.087 µm 3 ), cocci (0.155 µm 3 ) and vibrio-shaped cells (0.073 µm 3 ) showed much higher probabilities to be detected by CARD-FISH. These mor- photypes per se formed the highest contribution to total biomass, which explained the high detection efficiency of biomass with CARD-FISH. In addition, we determined the seasonal dynamics of morphotype distributions within 3 distinct phylogenetic lineages. Actinobacteria were predominately small rods and cocci, whereas bac- teria from the Cytophaga -Flavobacterium-Bacteroides group formed mainly large rods, cocci and filaments. Be- taproteobacteria showed the highest morphological vari- ability. Within all lineages, distinct spatio-temporal dy- namics of dominant morphotypes were observed. Thus, the approach presented here will allow for more detailed studies of the amount of carbon bound by different bac- terial taxa. This is of relevance as distinct lineages can contribute much more to total bacterial biomass than to total bacterioplankton abundance.