Springer protocols feed by Microbiology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Targeted Genomics of Flow Cytometrically Sorted Cultured and Uncultured Microbial Groups
High throughput sequencing of genetic material recovered from environmental samples (i.e., metagenomics) is becoming the method of choice for either medical or environmental genomic studies. However, the large amount of data and complexity of the sequenced “biomes” present challenges for teasing meaningful results out of the mass. Here, we describe a targeted genomic pipeline which uses fluorescence-activated cell sorting (FACS) in combination with multiple displacement amplification (MDA) of nucleic acids that allows to dissect a complex system into its component parts to facilitate high-quality single-cell, o...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Metagenomics Using Next-Generation Sequencing
Traditionally, microbial genome sequencing has been restricted to the small number of species that can be grown in pure culture [1]. The progressive development of culture-independent methods over the last 15 years now allows researchers to sequence microbial communities directly from environmental samples. This approach is commonly referred to as “metagenomics” or “community genomics”. However, the term metagenomics is applied liberally in the literature to describe any culture-independent analysis of microbial communities. Here, we define metagenomics as shotgun (“random”) sequencing o...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Stable Isotope Probing to Study Functional Components of Complex Microbial Ecosystems
This protocol presents a method of dissecting the DNA or RNA of key organisms involved in a specific biochemical process within a complex ecosystem. Stable isotope probing (SIP) allows the labelling and separation of nucleic acids from community members that are involved in important biochemical transformations, yet are often not the most numerically abundant members of a community. This pure culture-independent technique circumvents limitations of traditional microbial isolation techniques or data mining from large-scale whole-community metagenomic studies to tease out the identities and genomic repertoires of microorgani...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Bacterial Whole-Cell Biosensors for the Detection of Contaminants in Water and Soils
In this study, a standard method is described for the detection of contaminants and toxicity in real water and soil samples using Acinetobacter baylyi ADP1-based biosensors. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Single-Cell Raman Sorting
Single-cell Raman spectroscopy is a noninvasive and label-free technology for biochemical analysis of bacterial cells. A single-cell Raman spectrum functions as a metabolic “fingerprint” of an individual cell, which enables differentiation of cell types, physiological states, nutrient condition, and variable phenotypes. Raman tweezers combines single-cell Raman spectroscopy with optical laser tweezers to allow the identification and isolation of single living cells according to their Raman spectra. After cell sorting subsequent culturing and genomic sequencing has the potential to reveal totally new groups of m...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Visualization of Metabolic Properties of Bacterial Cells Using Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS)
NanoSIMS combines high-resolution imaging and mass spectrometry with simultaneous collection of up to seven different masses, providing an invaluable technique for determining the isotopic and elemental composition in microscopic target samples. It has been used in varying fields, from studying the elemental composition of mineral samples to tracking cell uptake of isotope-labelled substrates. In combination with in situ hybridization techniques, NanoSIMS offers a powerful method of linking metabolic capacity to phylogenetic identity in cell samples. Here, we describe methods and considerations for microbial sample prepara...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Analysis of Methanotroph Community Structure Using a pmoA-Based Microarray
The analysis of methanotroph community composition is relevant to studies of methane oxidation in a number of environments where methane is a significant carbon source. The development and application of a microarray targeting the particulate methane monooxygenase gene (pmoA) have allowed a high-throughput, semiquantitative analysis of the major methanotroph groups in a number of different environments. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Next Generation Barcode Tagged Sequencing for Monitoring Microbial Community Dynamics
Microbial identification using 16S rDNA variable regions has become increasingly popular over the past decade. The application of next-generation amplicon sequencing to these regions allows microbial communities to be sequenced in far greater depth than previous techniques, as well as allowing for the identification of unculturable or rare organisms within a sample. Multiplexing can be used to sequence multiple samples in tandem through the use of sample-specific identification sequences which are attached to each amplicon, making this a cost-effective method for large-scale microbial identification experiments. (Source: S...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Human Fecal Source Identification with Real-Time Quantitative PCR
We describe a real-time quantitative PCR (qPCR) method that targets a genetic marker of the human-associated Bacteroides dorei for identification of human fecal pollution in ambient water samples. The following protocol includes water sample collection, filtration, DNA isolation with a sample processing control, qPCR amplification with an internal amplification control, and quality control data analysis. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Profiling the Diversity of Microbial Communities with Single-Strand Conformation Polymorphism (SSCP)
Genetic fingerprinting techniques for microbial community analysis have evolved over the last decade into standard applications for efficient and fast differentiation of microbial communities based on their diversity. These techniques commonly analyze the diversity of PCR products amplified from extracted environmental DNA usually utilizing primers hybridizing to suspected conserved regions of the targeted genes. In comparison to the more commonly applied terminal restriction fragment length polymorphism (TRFLP) or denaturing gradient gel electrophoresis (DGGE) techniques, the here-described single-strand conformation poly...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Terminal Restriction Fragment Length Polymorphism (T-RFLP) Profiling of Bacterial 16S rRNA Genes
T-RFLP profiling is a very effective method for comparing many samples in an environmental microbiology study, because fingerprints of microbial diversity can be generated in a sensitive, reproducible, and cost-effective manner. This protocol describes the steps required to generate T-RFLP profiles of the dominant members of a bacterial community, by PCR amplification of the bacterial 16S rRNA genes and three restriction endonuclease digests to generate three different profiles for each sample. The generation of multiple profiles per sample provides enough information to confidently differentiate rich environmental bacteri...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Analysis of Community Dynamics in Environmental Samples Using Denaturing Gradient Gel Electrophoresis
Denaturing gradient gel electrophoresis (DGGE) is a culture-independent fingerprinting technique that allows for rapid comparative analysis of changes to microbial communities. 16S rRNA genes amplified from environmental samples can be separated based on their melting behavior in a denaturing gradient of urea and formamide. A fingerprint of the microbial community is generated with each band on the gel assumed to correspond to a different bacterial species. Community dynamics can then be assessed through statistical analysis of DGGE profiles and the sequencing of excised bands. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Biolog Phenotype MicroArrays for Phenotypic Characterization of Microbial Cells
Biolog Phenotype MicroArrays for microorganisms provide a high-throughput method for the global analysis of microbial growth phenotypes. Using a colorimetric reaction that is indicative of respiration, these microplate assays measure the response of an individual strain or microbial community to a large and diverse range of nutrients and chemicals. Phenotype MicroArrays have been used to study gene function and to improve genome annotation in single microorganisms and for physiological profiling of bacterial communities. The microplate system can be used to obtain a comprehensive overview of metabolic capability, or it can...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Quantitative PCR for Detection of mRNA and gDNA in Environmental Isolates
Quantitative PCR is used to gauge the abundance of specific nucleic acid species within purified samples. Due to its high sensitivity and minimal operation costs, this method is routinely applied in modern molecular bioscience laboratories. Nonetheless, all quantitative PCR experiments must include several carefully designed, yet simple, controls to ensure the reliability of the analyses. The aim of this chapter is to provide basic quantitative PCR methods, from primer design through data analysis, that are generally applicable to studies in microbiology. These methods allow the abundance of targeted RNA or DNA molecules t...
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
Rapid Extraction of PCR-Competent DNA from Recalcitrant Environmental Samples
Advances in sequencing technologies have made the investigation of microbial ecology and community dynamics more tractable. The critical first step in such analyses is the efficient and representative recovery of PCR-competent DNA from complex environmental samples. All extraction protocols contain inherent biases, meaning that choice of method involves compromise between various factors, including efficiency, yield, universality, and representative extraction. Here, details are given for a routine method used in our laboratory to extract DNA from soils, sediments, biofilms, roots, and fungi. (Source: Springer protocols feed by Microbiology)
Source: Springer protocols feed by Microbiology - January 1, 2014 Category: Microbiology Source Type: news
