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

Environmental dynamics as a structuring factor for microbial carbon utilization in a subtropical coastal lagoon

Laguna de Rocha belongs to a series of shallow coastal lagoons located along South America. It is periodically connected to the sea through a sand bar, exhibiting a hydrological cycle where physicochemical and biological gradients are rapidly established and destroyed. Its most frequent state is the separation of a Northern zone with low salinity, high turbidity and nutrient load, and extensive macrophyte growth, and a Southern zone with higher salinity and light penetration, and low nutrient content and macrophyte biomass. This zonation is reflected in microbial assemblages with contrasting abundance, activity, and community composition. The physicochemical conditions exerted a strong influence on community composition, and transplanted assemblages rapidly transformed to resembling the community of the recipient environment. Moreover, the major bacterial groups responded differently to their passage between the zones, being either stimulated or inhibited by the environmental changes, and exhibiting contrasting sensitivities to gradients. Addition of allochthonous carbon sources induced pronounced shifts in the bacterial communities, which in turn affected the microbial trophic web by stimulating heterotrophic flagellates and virus production. By contrast, addition of organic and inorganic nutrient sources (P or N) did not have significant effects. Altogether, our results suggest that (i) the planktonic microbial assemblage of this lagoon is predominantly carbon-limited, (ii) different bacterial groups cope differently with this constraint, and (iii) the hydrological cycle of the lagoon plays a key role for the alleviation or aggravation of bacterial carbon limitation. Based on these findings we propose a model of how hydrology affects the composition of bacterioplankton and of carbon processing in Laguna de Rocha.This might serve as a starting hypothesis for further studies about the microbial ecology of this lagoon, and of comparable transitional systems.Fil: Alonso, Cecilia. Universidad de la República; UruguayFil: Piccini, Claudia. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Unrein, Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Bertoglio, Florencia. Universidad de la República; UruguayFil: Conde, Daniel. Universidad de la República; UruguayFil: Pernthaler, Jakob. Universitat Zurich; Suiz

Antibiotic effects of three strains of chrysophytes (Ochromonas, Poterioochromonas) on freshwater bacterial isolates

We investigated the antibiotic effects of extracts of freeze-dried biomass and culture supernatants from the mixotrophic chrysophyte species Ochromonas danica, Poterioochromonas sp. strain DS, and Poterioochromonas malhamensis on bacterial strains isolated from lake water. Methanolic biomass extracts inhibited the growth of all tested strains, albeit to a different extent, whereas aqueous biomass extracts only affected bacteria of the genus Flectobacillus. The antibiotic action of supernatants from flagellate cultures could be mostly attributed to lipophilic substances, but the growth of bacteria affiliated with Flectobacillus and Sphingobium was also affected by hydrophilic compounds. A comparison of biomass extracts from light- and dark-adapted cultures of Poterioochromonas sp. strain DS showed that the growth-inhibiting factor was unrelated to chlorophyll derivatives. Supernatants from a dark-adapted, phagotrophically grown flagellate culture had stronger antibiotic effects and affected more bacterial strains than the supernatant from a light-adapted culture. Significant growth reduction of a Flectobacillus isolate was already induced by extremely low concentrations of lipophilic extracts from these supernatants. Our results show that metabolites of the studied flagellates − either released actively or during cell lysis − may selectively affect the growth of some aquatic bacteria even in very small doses and thus potentially affect microbial community composition. Moreover, the antibiotic potential of mixotrophic chrysophytes may change with their nutritional mod

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

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

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

Ecology and distribution of Thaumarchaea in the deep hypolimnion of Lake Maggiore

Ammonia-oxidizing Archaea (AOA) play an important role in the oxidation of ammonia in terrestrial, marine, and geothermal habitats, as confirmed by a number of studies specifically focused on those environments. Much less is known about the ecological role of AOA in freshwaters. In order to reach a high resolution at the Thaumarchaea community level, the probe MGI-535 was specifically designed for this study and applied to fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH) analysis. We then applied it to a fine analysis of diversity and relative abundance of AOA in the deepest layers of the oligotrophic Lake Maggiore, confirming previous published results of AOA presence, but showing differences in abundance and distribution within the water column without significant seasonal trends with respect to Bacteria. Furthermore, phylogenetic analysis of AOA clone libraries from deep lake water and from a lake tributary, River Maggia, suggested the riverine origin of AOA of the deep hypolimnion of the lake

Activity of metazoa governs biofilm structure formation and enhances permeate flux during Gravity-Driven Membrane (GDM) filtration

The impact of different feed waters in terms of eukaryotic populations and organic carbon content on the biofilm structure formation and permeate flux during Gravity-Driven Membrane (GDM) filtration was investigated in this study. GDM filtration was performed at ultra-low pressure (65 mbar) in dead-end mode without control of the biofilm formation. Different feed waters were tested (River water, pre-treated river water, lake water, and tap water) and varied with regard to their organic substrate content and their predator community. River water was manipulated either by chemically inhibiting all eukaryotes or by filtering out macrozoobenthos (metazoan organisms). The structure of the biofilm was characterized at the meso- and micro-scale using Optical Coherence Tomography (OCT) and Confocal Laser Scanning Microscopy (CLSM), respectively. Based on Total Organic Carbon (TOC) measurements, the river waters provided the highest potential for bacterial growth whereas tap water had the lowest. An increasing content in soluble and particulate organic substrate resulted in increasing biofilm accumulation on membrane surface. However, enhanced biofilm accumulation did not result in lower flux values and permeate flux was mainly influenced by the structure of the biofilm. Metazoan organisms (in particular nematodes and oligochaetes) built-up protective habitats, which resulted in the formation of open and spatially heterogeneous biofilms composed of biomass patches. In the absence of predation by metazoan organisms, a flat and compact biofilm developed. It is concluded that the activity of metazoan organisms in natural river water and its impact on biofilm structure balances the detrimental effect of a high biofilm accumulation, thus allowing for a broader application of GDM filtration. Finally, our results suggest that for surface waters with high particulate organic carbon (POC) content, the use of worms is suitable to enhance POC removal before ultrafiltration units

Widespread occurrence of an intranuclear bacterial parasite in vent and seep bathymodiolin mussels

Many parasitic bacteria live in the cytoplasm of multicellular animals, but only a few are known to regularly invade their nuclei. In this study, we describe the novel bacterial parasite "Candidatus Endonucleobacter bathymodioli" that invades the nuclei of deep-sea bathymodiolin mussels from hydrothermal vents and cold seeps. Bathymodiolin mussels are well known for their symbiotic associations with sulfur- and methane-oxidizing bacteria. In contrast, the parasitic bacteria of vent and seep animals have received little attention despite their potential importance for deep-sea ecosystems. We first discovered the intranuclear parasite "Ca. E. bathymodioli" in Bathymodiolus puteoserpentis from the Logatchev hydrothermal vent field on the Mid-Atlantic Ridge. Using primers and probes specific to "Ca. E. bathymodioli" we found this intranuclear parasite in at least six other bathymodiolin species from vents and seeps around the world. Fluorescence in situ hybridization and transmission electron microscopy analyses of the developmental cycle of "Ca. E. bathymodioli" showed that the infection of a nucleus begins with a single rod-shaped bacterium which grows to an unseptated filament of up to 20 μm length and then divides repeatedly until the nucleus is filled with up to 80 000 bacteria. The greatly swollen nucleus destroys its host cell and the bacteria are released after the nuclear membrane bursts. Intriguingly, the only nuclei that were never infected by "Ca. E. bathymodioli" were those of the gill bacteriocytes. These cells contain the symbiotic sulfur- and methane-oxidizing bacteria, suggesting that the mussel symbionts can protect their host nuclei against the parasite. Phylogenetic analyses showed that the "Ca. E. bathymodioli" belongs to a monophyletic clade of Gammaproteobacteria associated with marine metazoans as diverse as sponges, corals, bivalves, gastropods, echinoderms, ascidians and fish. We hypothesize that many of the sequences from this clade originated from intranuclear bacteria, and that these are widespread in marine invertebrates

Comparison of image analysis software packages in the assessment of adhesion of microorganisms to mucosal epithelium using confocal laser scanning microscopy

We have compared current image analysis software packages in order to find the most useful one for assessing microbial adhesion and inhibition of adhesion to tissue sections. We have used organisms of different sizes, the bacterium Helicobacter pylori and the yeast Candida albicans. Adhesion of FITC-labelled H. pylori and C. albicans was assessed by confocal microscopy. Four different Image analysis software packages, NIH-Image, IP Lab, Image Pro+, and Metamorph, were compared for their ability to quantify adhesion of the two organisms and several quantification methods were devised for each package. For both organisms, the dynamic range that could be detected by the software packages was 1×106?1×109 cells/ml. Of the four software packages tested, our results showed that Metamorph software, using our ?Region of Interest? method, with the software's ?Standard Area Method? of counting, was the most suitable for quantifying adhesion of both organisms because of its unique ability to separate clumps of microbial cells. Moreover, fewer steps were required. By pre-incubating H. pylori with the glycoconjugate Lewis b-HSA, an inhibition of binding of 48.8% was achieved using 250 ?g/ml Lewis b-HSA. The method we have devised using Metamorph software, provides a simple, quick and accurate way of quantifying adhesion and inhibition of adhesion of microbial cells to the epithelial surface of tissue sections. The method can be applied to organisms ranging in size from small bacteria to larger yeast cells