Detecting Bacterium that Cannot be Cultured Assignment

Detecting Bacterium that Cannot be Cultured Assignment

Detecting Bacterium that Cannot be Cultured Assignment
How can you detect the presence of bacterium that cannot be cultured? Explain your answer with specific examples

The first bacterial culture media were broths made either by infusion or by enzymatic digestion of meat from various sources. Originally developed by Spallanzani in the 18th century and then refined by Pasteur in the 19th century, these allowed the recovery of bacteria from human disease sites1,2. However, it was quickly realized that such broths would be likely to contain mixtures of micro-organisms, and Robert Koch saw the need for development of solid culture media that would allow the physical separation of bacterial colonies. He first tried aseptically divided potatoes. Material taken from infected lesions was spread across the potato and then incubated at body temperature. Following incubation, bacterial colonies were seen which could be subcultured to further potatoes to give pure cultures. Although successful, Koch observed that only a limited number of the micro-organisms present in the sample grew on the potato. This was probably the first recognition of the phenomenon of unculturability in vitro. Nevertheless, the success of the technique led to the use of solidifying agents such as gelatin and agar to create solid media from the broths developed by Pasteur and others. This advance led to the golden age of medical microbiology, in the last quarter of the 19th century, when many of the bacteria causing serious infections in man were identified. This tremendous success, however, probably led microbiologists to become complacent simply because so many important pathogenic bacteria could be cultured in vitro in this way.

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UNCULTURABILITY
We are grossly ignorant of bacterial life on earth. Environmental microbiologists estimate that less than 2% of bacteria can be cultured

in the laboratory. In the mouth we do rather better, with about 50% of the oral microflora being culturable3. For other body sites, the figure is unknown but is likely to be similar to that found in the mouth or higher. For example, the colonic microflora is suspected to be predominantly unculturable. It is therefore likely on numerical grounds alone that unculturable and therefore uncharacterized organisms are responsible for several oral and other human infections. A known instance is syphilis, caused by the spirochaete Treponema pallidum, which remains unculturable today.

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MOLECULAR IDENTIFICATION OF BACTERIA
It is only with the advent of molecular biology that techniques have become available for studying mixed bacterial communities in their entirety, without the biases of culture. The theoretical basis for the development of these methods came first from Zuckerandl and Pauling4, who suggested the use of biological macromolecules for the elucidation of evolutionary relationships among organisms. This idea, which developed into the branch of science known as molecular phylogeny, relies on the analysis of the DNA sequences of genes of common ancestry, or proteins themselves, in a range of organisms. Mathematical techniques are used to assess the similarity of these sequences and to construct phylogenetic trees which demonstrate the evolution of the whole organisms from which the DNA or proteins were isolated. In practice, ‘housekeeping’ genes are used for this purpose since they are widely distributed among different organisms and because their essential function has made them relatively conserved throughout evolution, allowing easy alignment of the sequences. The most widely used of these genes to date has been the small subunit (16S) ribosomal RNA gene5. At around 1500 base pairs in length this is both long enough to be informative and short enough to allow easy sequencing, particularly since the advent of automated DNA sequencers. Phylogenetic trees are constructed by first aligning sequences of the same gene from different organisms. Then the genetic distance between pairs of organisms in the dataset is calculated to give a matrix of similarities. This matrix is then further analysed by, for example, the neighbour-joining method in order to construct the phylogenetic tree or dendrogram6

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