We present an image analysis routine to determineud the contribution of distinct morphotypes to theud total abundance and biomass (carbon) of freshwater bacterioplankton and to the fraction of cells detected byud fluorescence in situ hybridization via catalyzed reporterud deposition (CARD-FISH). The method was tested onud bacterial assemblages from an alpine lake (Piburger See,ud Austria) at characteristic time points during the limnological year. Although on average 51% of 4’,6’-diamidino-2-phenylindole (DAPI)-stained objects were hybridized with the oligonucleotide probe EUB I-III, weud detected on average 80% of total biomass determinedud from DAPI staining. The assemblage was numericallyud dominated by cocci and rods <0.6 μm (mean cell volumeud = 0.024 μm3). Only a minor part of these morphotypesud could be hybridized (18 and 50%, respectively). In contrast,ud larger rods (0.087 μm3), cocci (0.155 μm3) andud vibrio-shaped cells (0.073 μm3) showed much higherud probabilities to be detected by CARD-FISH. These morphotypesud per se formed the highest contribution to totalud biomass, which explained the high detection efficiencyud of biomass with CARD-FISH. In addition, we determinedud the seasonal dynamics of morphotype distributionsud within 3 distinct phylogenetic lineages. Actinobacteriaud were predominately small rods and cocci, whereas bacteriaud from the Cytophaga–Flavobacterium–Bacteroidesud group formed mainly large rods, cocci and filaments. Betaproteobacteria showed the highest morphological variability. Within all lineages, distinct spatio-temporal dynamics of dominant morphotypes were observed. Thus,ud the approach presented here will allow for more detailedud studies of the amount of carbon bound by different bacterialud taxa. This is of relevance as distinct lineages canud contribute much more to total bacterial biomass than toud total bacterioplankton abundance