CiteULike: vrich's fcm

Applications of Flow Cytometry to Evolutionary and Population Biology

Paul Kron — 2008-05-10T20:36:23-00:00

Annual Review of Ecology, Evolution, and Systematics, Vol. 38, No. 1. (2007), pp. 847-876.

Flow cytometry, a method of rapidly characterizing optical properties of cells and cell components within individuals, populations, and communities, is advancing research in several areas of ecology, systematics, and evolutionary biology. Measuring the light emitted or scattered from cells or cell components, often in combination with specific stains, allows a multitude of physical and genetic attributes to be evaluated simultaneously and the resulting information to be rapidly processed. As a result, the technique has enabled large-scale comparative analyses of genome-size evolution, taxonomic identification and delineation, and studies of polyploids, reproductive biology, and experimental evolution. It is also being used to characterize the structure and composition of microbial communities. Here, we outline the nature of these contributions, as well as future applications, and provide an online summary of protocols and sampling methods.

Carbon-isotopic analysis of microbial cells sorted by flow cytometry

KM Eek — 2007-03-01T02:44:20-00:00

Geobiology, Vol. 5, No. 1. (March 2007), pp. 85-95.

Fate of Heterotrophic Microbes in Pelagic Habitats: Focus on Populations

Jakob Pernthaler — 2008-05-06T19:41:15-00:00

Microbiol. Mol. Biol. Rev., Vol. 69, No. 3. (1 September 2005), pp. 440-461.

Major biogeochemical processes in the water columns of lakes and oceans are related to the activities of heterotrophic microbes, e.g., the mineralization of organic carbon from photosynthesis and allochthonous influx or its transport to the higher trophic levels. During the last 15 years, cultivation-independent molecular techniques have substantially contributed to our understanding of the diversity of the microbial communities in different aquatic systems. In parallel, the complexity of aquatic habitats at a microscale has inspired research on the ecophysiological properties of uncultured microorganisms that thrive in a continuum of dissolved to particulate organic matter. One possibility to link these two aspects is to adopt a "Gleasonian" perspective, i.e., to study aquatic microbial assemblages in situ at the population level rather than looking at microbial community structure, diversity, or function as a whole. This review compiles current knowledge about the role and fate of different populations of heterotrophic picoplankton in marine and inland waters. Specifically, we focus on a growing suite of techniques that link the analysis of bacterial identity with growth, morphology, and various physiological activities at the level of single cells. An overview is given of the potential and limitations of methodological approaches, and factors that might control the population sizes of different microbes in pelagic habitats are discussed. 10.1128/MMBR.69.3.440-461.2005

Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry

Jeremy Lenaerts — 2008-05-06T18:50:55-00:00

Appl. Environ. Microbiol., Vol. 73, No. 6. (15 March 2007), pp. 2020-2023.

Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry. 10.1128/AEM.01718-06

Extreme spatial variability in marine picoplankton and its consequences for interpreting Eulerian time-series

Adrian Martin — 2008-05-06T01:04:14-00:00

Biology Letters, Vol. 1, No. 3. (2005), pp. 366-369.

A high-resolution mesoscale spatial survey of picoplankton in the Celtic Sea, using flow cytometry, reveals cell concentrations of Synechococcus spp. cyanobacteria and heterotrophic bacteria that vary up to 50-fold over distances as short as 12km. Furthermore, the range of abundances is comparable to that typically found on seasonal scales at a single location. Advection of such spatial variability through a time-series site would therefore constitute a major source of ‘error’. Consequently, attempts to model and to investigate the ecology of these globally important organisms in situ must take into account and quantify the hitherto ignored local spatial variability as a matter of necessity.

Flow Sorting of Marine Bacterioplankton after Fluorescence In Situ Hybridization

Raju Sekar — 2006-12-08T14:18:48-00:00

Appl. Environ. Microbiol., Vol. 70, No. 10. (1 October 2004), pp. 6210-6219.

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.

Niche Partitioning Among Prochlorococcus Ecotypes Along Ocean-Scale Environmental Gradients

Zackary Johnson — 2006-04-18T15:59:09-00:00

Science, Vol. 311, No. 5768. (24 March 2006), pp. 1737-1740.

Prochlorococcus is the numerically dominant phytoplankter in the oligotrophic oceans, accounting for up to half of the photosynthetic biomass and production in some regions. Here, we describe how the abundance of six known ecotypes, which have small subunit ribosomal RNA sequences that differ by less than 3%, changed along local and basin-wide environmental gradients in the Atlantic Ocean. Temperature was significantly correlated with shifts in ecotype abundance, and laboratory experiments confirmed different temperature optima and tolerance ranges for cultured strains. Light, nutrients, and competitor abundances also appeared to play a role in shaping different distributions. 10.1126/science.1118052

Flow cytometry for microbial sensing in environmental sustainability applications: current status and future prospects

Cyndee Gruden — 2008-05-05T14:44:00-00:00

FEMS Microbiology Ecology, Vol. 49, No. 1. (2004), pp. 37-49.

Abstract Practical and accurate microbial assessment of environmental systems is predicated on the detection and quantification of various microbial parameters in complex matrices. Traditional growth-based assays, considered to be both slow and biased, are increasingly being replaced by optical detection methods such as flow cytometry. Flow cytometry (FCM) offers high-speed multi-parametric data acquisition, compatibility with current molecular-based microbial detection technologies, and is a proven technology platform. The unique technical properties of flow cytometry have allowed the discrimination of bacteria based on nucleic acid staining, microbial identification based on genomic and immunologic characteristics, and determination of cell viability. For this technology to achieve the ultimate goal of monitoring the microbial ecology of distributed systems, it will be necessary to develop a fully functional, low cost, and networkable microsystem platform capable of rapid detection of multiple species of microorganisms simultaneously under realistic environmental conditions. One such microsystem, miniaturized and integrated in accordance with recent advances in micro-electro-mechanical systems technology, is named the Micro Integrated Flow Cytometer. This manuscript is a minireview of the current status and future prospects for environmental application of flow cytometry in general, and micro-flow cytometry in particular.

Flow cytometric enumeration of DNA-stained oceanic planktonic protists

Zubkov — 2007-03-30T06:35:22-00:00

Journal of Plankton Research, Vol. 29, No. 1. (1 January 2007), pp. 79-86.

The emergence of automated high-frequency flow cytometry: revealing temporal and spatial phytoplankton variability

Melilotus Thyssen — 2008-04-28T01:48:54-00:00

J. Plankton Res., Vol. 30, No. 3. (1 March 2008), pp. 333-343.

Phytoplankton observation is the product of a number of trade-offs related to sampling processes, required level of diversity and size spectrum analysis capabilities of the techniques involved. Instruments combining the morphological and high-frequency analysis for phytoplankton cells are now available. This paper presents an application of the automated high-resolution flow cytometer Cytosub as a tool for analysing phytoplanktonic cells in their natural environment. High resolution data from a temporal study in the Bay of Marseille (analysis every 30 min over 1 month) and a spatial study in the Southern Indian Ocean (analysis every 5 min at 10 knots over 5 days) are presented to illustrate the capabilities and limitations of the instrument. Automated high-frequency flow cytometry revealed the spatial and temporal variability of phytoplankton in the size range 1-[~]50 microm that could not be resolved otherwise. Due to some limitations (instrumental memory, volume analysed per sample), recorded counts could be statistically too low. By combining high-frequency consecutive samples, it is possible to decrease the counting error, following Poisson's law, and to retain the main features of phytoplankton variability. With this technique, the analysis of phytoplankton variability combines adequate sampling frequency and effective monitoring of community changes. 10.1093/plankt/fbn005