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We present an image analysis routine to determine\udthe contribution of distinct morphotypes to the\udtotal abundance and biomass (carbon) of freshwater bacterioplankton and to the fraction of cells detected by\udfluorescence in situ hybridization via catalyzed reporter\uddeposition (CARD-FISH). The method was tested on\udbacterial assemblages from an alpine lake (Piburger See,\udAustria) 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, we\uddetected on average 80% of total biomass determined\udfrom DAPI staining. The assemblage was numerically\uddominated by cocci and rods <0.6 μm (mean cell volume\ud= 0.024 μm3). Only a minor part of these morphotypes\udcould be hybridized (18 and 50%, respectively). In contrast,\udlarger rods (0.087 μm3), cocci (0.155 μm3) and\udvibrio-shaped cells (0.073 μm3) showed much higher\udprobabilities to be detected by CARD-FISH. These morphotypes\udper se formed the highest contribution to total\udbiomass, which explained the high detection efficiency\udof biomass with CARD-FISH. In addition, we determined\udthe seasonal dynamics of morphotype distributions\udwithin 3 distinct phylogenetic lineages. Actinobacteria\udwere predominately small rods and cocci, whereas bacteria\udfrom the Cytophaga–Flavobacterium–Bacteroides\udgroup 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,\udthe approach presented here will allow for more detailed\udstudies of the amount of carbon bound by different bacterial\udtaxa. This is of relevance as distinct lineages can\udcontribute much more to total bacterial biomass than to\udtotal bacterioplankton abundance

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