We describe an approach to sort cells from coastal North Sea bacterioplankton by flow cytometry after in situ hybridization with rRNA-targeted horseradish peroxidase-labeled oligonucleotide probes and catalyzed fluorescent reporter deposition (CARD-FISH). In a sample from spring 2003 >90% of the cells were detected by CARD-FISH with a bacterial probe (EUB338). Approximately 30% of the microbial assemblage was affiliated with the Cytophaga-Flavobacterium lineage of the Bacteroidetes (CFB group) (probe CF319a), and almost 10% was targeted by a probe for the ␤-proteobacteria (probe BET42a). A protocol was optimized to detach cells hybridized with EUB338, BET42a, and CF319a from membrane filters (recovery rate, 70%) and to sort the cells by flow cytometry. The purity of sorted cells was >95%. 16S rRNA gene clone libraries were constructed from hybridized and sorted cells (S-EUB, S-BET, and S-CF libraries) and from unhybridized and unsorted cells (UNHYB library). Sequences related to the CFB group were significantly more frequent in the S-CF library (66%) than in the UNHYB library (13%). No enrichment of ␤-proteobacterial sequence types was found in the S-BET library, but novel sequences related to Nitrosospira were found exclusively in this library. These bacteria, together with members of marine clade OM43, represented >90% of the ␤-proteobacteria in the water sample, as determined by CARD-FISH with specific probes. This illustrates that a combination of CARD-FISH and flow sorting might be a powerful approach to study the diversity and potentially the activity and the genomes of different bacterial populations in aquatic habitats.

The objective of this study was to investigate the autoaggregation, coaggregation and biofilm formation of four bacteria namely Sphingobium, Xenophilus, Methylobacterium and Rhodococcus isolated from drinking water. Auto and coaggregation studies were performed by both qualitative (DAPI staining) and semi-quantitative (visual coaggregation) methods and biofilms produced by either pure or dual-cultures were quantified by crystal violet method. Results from the semi-quantitative visual aggregation method did not show any immediate auto or coaggregation, which was confirmed by the 4′,6 diamidino-2-phenylindole (DAPI) staining method. However, after 2 hours, Methylobacterium showed the highest autoaggregation of all four isolates. The Methylobacterium combinations showed highest coaggregation between dual species at extended period of times (72 hours). Biofilm formation by pure cultures was negligible in comparison to the quantity of biofilm produced by dual-species biofilms. The overall results show that coaggregation of bacteria isolated from drinking water was mediated by species-specific and time-dependent interactions with a synergistic type of biofilm formation. The results of this study are therefore a useful step in assisting the development of potential control strategies by identifying specific bacteria that promote aggregation or biofilm formation in drinking water distribution systems.

No abstract

Aims:  To determine the spatial and temporal variability in the abundance, structure and composition of planktonic bacterial assemblages sampled from a small, looped water distribution system and to interpret results with respect to hydraulic conditions. Methods and Results:  Water samples were collected from five sampling points, twice a day at 06:00 h and 09:00 h on a Monday (following low weekend demand) and a Wednesday (higher midweek demand). All samples were fully compliant with current regulated parameter standards. This study did not show obvious changes in bacterial abundance (DAPI count) or community structure Denaturing gradient gel electrophoresis analysis with respect to sample site and hence to water age; however, the study did show temporal variability with respect to both sampling day and sample times. Conclusions:  Data suggests that variations in the bacterial assemblages may be associated with the local system hydraulics: the bacterial composition and numbers, over short durations, are governed by the interaction of the bulk water and the biofilm influenced by the hydraulic conditions. Significance and Impact of the Study:  This study demonstrates general stability in bacterial abundance, community structure and composition within the system studied. Trends and patterns supporting the transfer of idealized understanding to the real world were evident. Ultimately, such work will help to safeguard potable water quality, fundamental to public health.

No abstract