Multi-spot live-image autofocusing for high-throughput microscopy of fluorescently stained bacteria

Screening by automated high-throughput microscopy has become a valuable research tool. An essential component of such systems is the autonomous acquisition of focused images. Here we describe the implementation of a high-precision autofocus routine for imaging of fluorescently stained bacteria on a commercially available microscope. We integrate various concepts and strategies that together substantially enhance the performance of autonomous image acquisition. These are (i) nested focusing in brightfield and fluorescence illumination, (ii) autofocusing by continuous life-image acquisition during movement in z-direction rather than at distinct z-positions, (iii) assessment of the quality and topology of a field of view (FOV) by multi-spot focus measurements and (iv) acquisition of z-stacks and application of an extended depth of field algorithm to compensate for FOV unevenness. The freely provided program and documented source code allow ready adaptation of the here presented approach to various platforms and scientific questions

A Novel Algorithm for the Determination of Bacterial Cell Volumes That is Unbiased by Cell Morphology

The determination of cell volumes and biomass offers a means of comparing the standing stocks of auto- and heterotrophic microbes of vastly different sizes for applications including the assessment of the flux of organic carbon within aquatic ecosystems. Conclusions about the importance of particular genotypes within microbial communities (e.g., of filamentous bacteria) may strongly depend on whether their contribution to total abundance or to biomass is regarded. Fluorescence microscopy and image analysis are suitable tools for determining bacterial biomass that moreover hold the potential to replace labor-intensive manual measurements by fully automated approaches. However, the current approaches to calculate bacterial cell volumes from digital images are intrinsically biased by the models that are used to approximate the morphology of the cells. Therefore, we developed a generic contour based algorithm to reconstruct the volumes of prokaryotic cells from two-dimensional representations (i.e., microscopic images) irrespective of their shape. Geometric models of commonly encountered bacterial morphotypes were used to verify the algorithm and to compare its performance with previously described approaches. The algorithm is embedded in a freely available computer program that is able to process both raw (8-bit grayscale) and thresholded (binary) images in a fully automated manner

Flow sorting of marine bacterioplankton after fluorescence in situ hybridization

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 beta-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 beta-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 beta-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

A Novel Algorithm for the Determination of Bacterial Cell Volumes That is Unbiased by Cell Morphology

The determination of cell volumes and biomass offers a means of comparing the standing stocks of auto- and heterotrophic microbes of vastly different sizes for applications including the assessment of the flux of organic carbon within aquatic ecosystems. Conclusions about the importance of particular genotypes within microbial communities (e.g., of filamentous bacteria) may strongly depend on whether their contribution to total abundance or to biomass is regarded. Fluorescence microscopy and image analysis are suitable tools for determining bacterial biomass that moreover hold the potential to replace labor-intensive manual measurements by fully automated approaches. However, the current approaches to calculate bacterial cell volumes from digital images are intrinsically biased by the models that are used to approximate the morphology of the cells. Therefore, we developed a generic contour based algorithm to reconstruct the volumes of prokaryotic cells from two-dimensional representations (i.e., microscopic images) irrespective of their shape. Geometric models of commonly encountered bacterial morphotypes were used to verify the algorithm and to compare its performance with previously described approaches. The algorithm is embedded in a freely available computer program that is able to process both raw (8-bit grayscale) and thresholded (binary) images in a fully automated manne

Scent of danger: floc formation by a freshwater bacterium is induced by supernatants from a predator-prey coculture

We investigated predator-prey interactions in a model system consisting of the bacterivorous flagellate Poterioochromonas sp. strain DS and the freshwater bacterium Sphingobium sp. strain Z007. This bacterial strain tends to form a subpopulation of grazing-resistant microscopic flocs, presumably by aggregation. Enhanced formation of such flocs could be demonstrated in static batch culture experiments in the presence of the predator. The ratio of aggregates to single cells reached >0.1 after 120 h of incubation in an oligotrophic growth medium. The inoculation of bacteria into supernatants from cocultures of bacteria and flagellates (grown in oligotrophic or in rich media) also resulted in a substantially higher level of floc formation than that in supernatants from bacterial monocultures only. After separation of supernatants on a C(18) cartridge, the aggregate-inducing activity could be assigned to the 50% aqueous methanolic fraction, and further separation of this bioactive fraction could be achieved by high-pressure liquid chromatography. These results strongly suggest the involvement of one or several chemical factors in the induction of floc formation by Sphingobium sp. strain Z007 that are possibly released into the surrounding medium by flagellate grazing

Diverse syntrophic partnerships from deep-sea methane vents revealed by direct cell capture and metagenomics

Microorganisms play a fundamental role in the cycling of nutrients and energy on our planet. A common strategy for many microorganisms mediating biogeochemical cycles in anoxic environments is syntrophy, frequently necessitating close spatial proximity between microbial partners. We are only now beginning to fully appreciate the diversity and pervasiveness of microbial partnerships in nature, the majority of which cannot be replicated in the laboratory. One notable example of such cooperation is the interspecies association between anaerobic methane oxidizing archaea (ANME) and sulfate-reducing bacteria. These consortia are globally distributed in the environment and provide a significant sink for methane by substantially reducing the export of this potent greenhouse gas into the atmosphere. The interdependence of these currently uncultured microbes renders them difficult to study, and our knowledge of their physiological capabilities in nature is limited. Here, we have developed a method to capture select microorganisms directly from the environment, using combined fluorescence in situ hybridization and immunomagnetic cell capture. We used this method to purify syntrophic anaerobic methane oxidizing ANME-2c archaea and physically associated microorganisms directly from deep-sea marine sediment. Metagenomics, PCR, and microscopy of these purified consortia revealed unexpected diversity of associated bacteria, including Betaproteobacteria and a second sulfate-reducing Deltaproteobacterial partner. The detection of nitrogenase genes within the metagenome and subsequent demonstration of 15N2 incorporation in the biomass of these methane-oxidizing consortia suggest a possible role in new nitrogen inputs by these syntrophic assemblages

Community structure, picoplankton grazing and zooplankton control of heterotrophic nanoflagellates in a eutrophic reservoir during the summer phytoplankton maximum

An intensive 5 wk study was conducted to investigate the role of protists, especially heterotrophic nanoflagellates (HNF), in microbial food webs during the summer phytoplankton bloom in the epilimnion and metalimnion of the eutrophic Rimov reservoir (South Bohemia, Czech Republic). On average, protists consumed similar to 90% of bacterial production in both layers. The community composition of HNF and the relative importance of different HNF groups as picoplankton consumers were determined. Small HNF (<8 mu m), as chrysomonads, bodonids and choanoflagellates, usually accounted for <30% of total HNF biomass but numerically dominated the community in both layers. They consumed most of (similar to 70 to 85 %) the bacterioplankton as well as autotrophic picoplankton (APP, exclusively cyanobacteria) production in the reservoir, with the rest consumed by ciliates. Both ciliates and HNF had higher clearance rates on APP than on bacteria and their grazing was likely responsible for a sharp decrease in APP abundance (from 3-4 x 10(5) to <2 x 10(3) ml(-1)) and a very constant size structure of bacterioplankton in which short rods in the size class of 0.4 to 0.8 mu m constituted 55 to 80 % of the total bacterial biomass in both layers. The proportion of HNF to total picoplankton biomass in the epilimnion indicated that the picoplankton biomass was sufficiently high to support HNF growth for most of the study. Uptake of picoplankton by less numerous, but larger, HNF (kathablepharids, Goniomonas sp., and Streptomonas sp.) was negligible, while their biomass, especially in the metalimnion, exceeded that of small HNF and the total biomass of picoplankton. This suggested that food items other than picoplankton were consumed to meet their carbon requirements. Analyzing potential bottom-up and top-down factors controlling HNF numbers and biomass, we did not find a tight relationship between HNF and the concentration of bacteria and chlorophyll. Variability of HNF abundance and biomass in the epilimnion could largely be explained by cladocerans or by pooled abundances of all potential crustacean consumers of HNF. In the metalimnion, the mean cell volume of HNF was positively linked to chlorophyll but negatively to the abundance of Cyclopidae and to the pooled abundances of Ceriodaphnia quadrangula and Diaphanosoma brachyurum

Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity

Extensive carbon isotopic heterogeneity among methane seep microbiota

To assess and study the heterogeneity of δ^(13)C values for seep microorganisms of the Eel River Basin, we studied two principally different sample sets: sediments from push cores and artificial surfaces colonized over a 14 month in situ incubation. In a single sediment core, the δ^(13)C compositions of methane seep-associated microorganisms were measured and the relative activity of several metabolisms was determined using radiotracers. We observed a large range of archaeal δ^(13)C values (> 50‰) in this microbial community. The δ^(13)C of ANME-1 rods ranged from −24‰ to −87‰. The δ^(13)C of ANME-2 sarcina ranged from −18‰ to −75‰. Initial measurements of shell aggregates were as heavy as −19.5‰ with none observed to be lighter than −57‰. Subsequent measurements on shell aggregates trended lighter reaching values as ^(13)C-depleted as −73‰. The observed isotopic trends found for mixed aggregates were similar to those found for shell aggregates in that the initial measurements were often enriched and the subsequent analyses were more ^(13)C-depleted (with values as light as −56‰). The isotopic heterogeneity and trends observed within taxonomic groups suggest that ANME-1 and ANME-2 sarcina are capable of both methanogenesis and methanotrophy. In situ microbial growth was investigated by incubating a series of slides and silicon (Si) wafers for 14 months in seep sediment. The experiment showed ubiquitous growth of bacterial filaments (mean δ^(13)C = −38 ± 3‰), suggesting that this bacterial morphotype was capable of rapid colonization and growth