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Until recently, knowledge of ruminant gut microbiology was primarily obtained using classical culture based techniques, which probably only account for 10 to 20% of the rumen microbial population. New gene-based technologies can now be employed to examine microbial diversity through the use of small sub-unit ribosomal DNA analysis (e.g. 16S rDNA) and to understand the function of complex microbial ecosystems in the rumen through metagenomic analysis. These technologies have the potential to revolutionize the understanding of rumen function and will overcome the limitations of classical based techniques, including isolation and taxonomic identification of strains important to efficient rumen function and better understanding of the roles of microorganisms in relation to achieving high productivity and decreasing environmental pollutants. This book presents a comprehensive up-to-date account of the methodologies and protocols for conventional and modern molecular techniques that are currently in use for studying the gut microbial ecology of ruminants. Each chapter has been contributed by experts in the field and methods have been presented in a recipe-like format designed for direct practical use in the laboratory and also to provide insight into the most appropriate techniques, their applications and the type of information that could be expected. The techniques and procedures described are also relevant and adaptable to other gastrointestinal ecosystems and the microbiology of anaerobic environments in general. This manual will `demystify' the methods in molecular microbial ecology for readers who are novice in the field but are excited by the prospects of the technology. It would also be invaluable for the experienced workers striving for giving new dimension to their research - expanding the work in other fields and initiating cross-cutting activities.
Foreword; Introduction; Editorial Note; Part 1. Designing in-vivo Microbial Ecology Studies; 1.1. Experimental designs for rumen microbiology, Adrian R. Egan; Part 2. Classical Methods for Isolation, Enumeration, Cultivation and Functional Assays of Rumen Microbes; 2.1. Rumen bacteria, Christopher S. McSweeney, Stuart E. Denman and Roderick I. Mackie; 2.2. Bacteriophages, Athol V. Klieve. 2.3. Methanogenic archaea, Keith N. Joblin; 2.4. Anaerobic fungi, Michael K. Theodorou, Jayne Brookman and Anthony P.J. Trinci; 2.5. Ciliate protozoa, Burk A. Dehority; Part 3. PCR-Based Methods for Analysis of Populations and Gene Expression; 3.1. Nucleic acid extraction, oligonucleotide probes and PCR methods, Zhongtang Yu and Robert J. Forster; 3.2. Quantitative (real-time) PCR, Stuart E. Denman and Christopher S. McSweeney; Part 4. Molecular Fingerprinting Techniques for Genotypic Analysis of Pure Cultures and Microbial Communities; 4.1. Denaturing gradient gel electrophoresis, Svetlana A. Kocherginskaya, Isaac K.O. Cann and Roderick I. Mackie; 4.2. Bacteriophage populations, Athol V. Klieve and Rosalind A. Gilbert; 4.3. Anaerobic fungal populations, Jayne L. Brookman and Matthew J. Nicholson; 4.4. RAPD, RFLP, T-RFLP, AFLP, RISA, Stuart E. Denman, Makoto Mitsumori and Christopher S. McSweeney; Part 5. DNA Clone Libraries of Microbial Communities; 5.1. 16S/18S ribosomal DNA clone library analysis of rumen microbial diversity, Andre-Denis G. Wright, Kiyoshi Tajima and Rustam I. Aminov; Part 6. Use of Small Subunit Ribosomal RNA Directed Oligonucleotide Probes for Microbial Population Studies; 6.1. Northern blot analysis to investigate the abundance of micro-organisms, Denis O. Krause; 6.2. Whole cell probing with fluorescently labelled probes for in situ analysis of microbial populations, Linda L. Blackall; 6.3. Combined fluorescence in situ hybridization and microautoradiography (FISH-MAR), Maneesha P. Ginige; Part 7. Genomic Analysis of Microbial Ecosystems; 7.1. Metagenomic analysis of the microbiomes in ruminants and other herbivores Mark Morrison, Sarah E. Adams, Karen E. Nelson and Graeme T. Attwood; Chapterwise Keywords; Keyword Index.