The bacterial colonies are then identified based on morphological and biochemical characteristics. These techniques are simple, cheap, readily available, and allow specific detection and semiquantitative estimation of several bacterial groups such as Bacteroides spp., Eubacterium spp., Bifidobacterium spp., Lactobacilli, and Clostridium spp. However, several bacterial species, in particular strict anaerobes, are quite fastidious, and do not grow on the available culture media under usual laboratory conditions. Data from newer molecular methods indicate that culture methods cannot detect nearly 80% of the bacterial species resident in the human gut and thus

underestimate the diversity of gut flora.[7] On the contrary, different strains of the same bacterial species may at www.selleckchem.com/products/pci-32765.html times vary in their characteristics, providing a false sense of diversity.[8] These techniques depend on diversity in the sequence of the bacterial 16S ribosomal RNA (rRNA) gene, which is present in all bacteria. It is 1.5 kilobase in length and has some regions that are strongly conserved and others that are highly variable. This

provides an appropriate balance of conservation among larger phylogenetic CDK inhibitor groups and sufficient variability to distinguish between different species.[9] Several techniques, each with its own advantages and limitations, have been developed to exploit this variability in 16S rRNA gene for the study of gut

microbiota. In this technique, the entire 16S rRNA gene is amplified and sequenced; learn more the bacterial species is then determined by comparing the sequence against one of several databases that contain sequences of this gene in various bacterial species. This technique provides the most accurate method of bacterial identification; however, it is costly, time consuming, and applicable primarily to pure bacterial cultures. It cannot be applied to complex bacterial mixtures such as the gut microbiota, except after successful culture of bacteria or cloning of bacterial DNA into a vector, followed by sequencing of DNA from several individual colonies or of several clones; this however is very costly. Also, these techniques introduce a bias because of failure of several bacterial species to grow or of their DNAs to be cloned. Several newer-generation sequencing platforms have become available in the last few years. These instruments allow rapid, high-throughput sequencing (nearly 25 million bases at 99% or better accuracy in a single 4-h run) at a much lower cost than traditional sequencing and have a particular advantage of ability to sequence individual molecules in a mixture of several nucleic acids.[10] These techniques target the hypervariable segments of the 16S rRNA gene. In brief, primers based on conserved regions surrounding a hypervariable region are used to amplify nucleic acids extracted from a bacterial mixture.