Culture-based techniques have traditionally been used to characterize the dominant and indicator microbial populations involved in the manufacture, ripening and safety of dairy products. The identification of microbes by their phenotypic traits and through the use of biochemical tests has also provided microbiological descriptions of dairy ecosystems. However, these techniques are expensive, labour intensive and require previous knowledge of each microbial species’ nutritional and growth requirements. In recent decades, culture methods have gradually been supplemented by an array of culture-independent molecular techniques, mostly based on comparisons of the structure of the 16S rRNA gene. These techniques were first developed to study the microbial diversity of complex environments. Molecular techniques overcome the limitations associated with conventional culture-based methods, including their low sensitivity, their inability to detect non-cultivatable bacteria and unknown species, their slow turnaround time, and their poor reproducibility. Using molecular techniques, microorganisms can be identified, quantified, and their phylogenetic relationships predicted. The choice of a molecular technique depends on the question being addressed. Cloning and the analysis of gene sequences might be used for the identification of microbiota at the species or subspecies level. Microbial community structure can readily be analysed by population fingerprinting techniques, such as denaturing gradient gel electrophoresis (DGGE), thermal gradient gel electrophoresis (TGGE), single-strand conformation polymorphism (SSCP), or terminal restriction fragment length polymorphism (T-RFLP), while dot blot hybridisation or fluorescent in situ hybridisation (FISH) can measure the abundance of particular species or taxonomic groups. In the near future, the emerging technologies of quantitative real time PCR (Q-PCR) and microarray hybridisation systems will be optimised and will likely be universally used.
Culture-independent microbial techniques in dairy microbiology: the state of the art.
MARZOTTO, Marta;TORRIANI, Sandra
2008-01-01
Abstract
Culture-based techniques have traditionally been used to characterize the dominant and indicator microbial populations involved in the manufacture, ripening and safety of dairy products. The identification of microbes by their phenotypic traits and through the use of biochemical tests has also provided microbiological descriptions of dairy ecosystems. However, these techniques are expensive, labour intensive and require previous knowledge of each microbial species’ nutritional and growth requirements. In recent decades, culture methods have gradually been supplemented by an array of culture-independent molecular techniques, mostly based on comparisons of the structure of the 16S rRNA gene. These techniques were first developed to study the microbial diversity of complex environments. Molecular techniques overcome the limitations associated with conventional culture-based methods, including their low sensitivity, their inability to detect non-cultivatable bacteria and unknown species, their slow turnaround time, and their poor reproducibility. Using molecular techniques, microorganisms can be identified, quantified, and their phylogenetic relationships predicted. The choice of a molecular technique depends on the question being addressed. Cloning and the analysis of gene sequences might be used for the identification of microbiota at the species or subspecies level. Microbial community structure can readily be analysed by population fingerprinting techniques, such as denaturing gradient gel electrophoresis (DGGE), thermal gradient gel electrophoresis (TGGE), single-strand conformation polymorphism (SSCP), or terminal restriction fragment length polymorphism (T-RFLP), while dot blot hybridisation or fluorescent in situ hybridisation (FISH) can measure the abundance of particular species or taxonomic groups. In the near future, the emerging technologies of quantitative real time PCR (Q-PCR) and microarray hybridisation systems will be optimised and will likely be universally used.
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