Detection of Viable Organisms Using Molecular Techniques
from Paul A. Rochelle, Anne K. Camper, Andreas Nocker and Mark Burr in Environmental Microbiology: Current Technology and Water Applications
The ultimate measure of microbial viability and biological activity is growth in some form of culture system. Unfortunately, due to many limitations, growth is usually not the most sensitive or rapid detection method. Many molecular-based tools are available for assessing viability and functional gene expression, and have applications for specific microbes in environmental samples. Methods include fluorescent nucleic acid binding dyes, enzymatic conversion of substrates to fluorescent compounds (often in conjunction with nucleic acid-based methods), various techniques based on amplification and detection of nucleic acids, nucleic acid amplification linked to biosensors and microarray detection platforms, detection and characterization of proteins, and molecular detection coupled with culturing.
Further reading:
The ultimate measure of microbial viability and biological activity is growth in some form of culture system. Unfortunately, due to many limitations, growth is usually not the most sensitive or rapid detection method. Many molecular-based tools are available for assessing viability and functional gene expression, and have applications for specific microbes in environmental samples. Methods include fluorescent nucleic acid binding dyes, enzymatic conversion of substrates to fluorescent compounds (often in conjunction with nucleic acid-based methods), various techniques based on amplification and detection of nucleic acids, nucleic acid amplification linked to biosensors and microarray detection platforms, detection and characterization of proteins, and molecular detection coupled with culturing.
Further reading:
Identity of Single Microbial Cells
from Daniel S. Read and Andrew S. Whiteley in Environmental Microbiology: Current Technology and Water Applications
Linking both identity and function within microbial communities has long been seen as essential for understanding the role that bacteria play in the environment. Techniques based on the study of single microbial cells offer a unique approach that provides information about heterogeneity within populations, and the role of spatial organization within the environment. Various single-cell techniques are currently in use for the study of microbial ecology, an important one being Raman spectroscopy. This technique can be used for studying different features of microbial systems. Raman spectroscopy can be used in combination with Fluorescence in situ Hybridization (FISH) and Stable Isotope Probing (SIP), which together can be utilized to gain an insight into the identity and function of single bacterial cells in situ.
Further reading:
Linking both identity and function within microbial communities has long been seen as essential for understanding the role that bacteria play in the environment. Techniques based on the study of single microbial cells offer a unique approach that provides information about heterogeneity within populations, and the role of spatial organization within the environment. Various single-cell techniques are currently in use for the study of microbial ecology, an important one being Raman spectroscopy. This technique can be used for studying different features of microbial systems. Raman spectroscopy can be used in combination with Fluorescence in situ Hybridization (FISH) and Stable Isotope Probing (SIP), which together can be utilized to gain an insight into the identity and function of single bacterial cells in situ.
Further reading:
Amoebae as a Tool
from Julia Lienard and Gilbert Greub in Environmental Microbiology: Current Technology and Water Applications
Obligate intracellular microorganisms are unculturable by classic axenic culture methods. As a result they have largely been overlooked, despite many being significant human and animal pathogens. Resistance of amoeba-resisting microorganisms (ARM) to amoebal destruction may predict ability to also resist mammalian macrophages, which are somehow similar to amoebae and represent one of the first cellular immune defenses in mammals. Thus, general approaches have been described for the growth of strict intracellular microorganisms, using amoebae as hosts in a cell culture system. Such an approach has been shown to be advantageous, since amoebal co-culture will selectively grow microorganisms that resist these professional phagocytes. An alternative approach for the isolation of novel ARM is also available, which requires the isolation of new amoebal strains by amoebal enrichment on a suitable prey (such as Escherichia coli), and then to search for intra-amoebal microorganisms within the isolated amoebae. Once new potentially pathogenic ARM has been isolated, one should then further assess the potential infectivity of these intracellular microorganisms. The application of macrophages, as an in vitro model to test microbial virulence is also possible.
Further reading:
Obligate intracellular microorganisms are unculturable by classic axenic culture methods. As a result they have largely been overlooked, despite many being significant human and animal pathogens. Resistance of amoeba-resisting microorganisms (ARM) to amoebal destruction may predict ability to also resist mammalian macrophages, which are somehow similar to amoebae and represent one of the first cellular immune defenses in mammals. Thus, general approaches have been described for the growth of strict intracellular microorganisms, using amoebae as hosts in a cell culture system. Such an approach has been shown to be advantageous, since amoebal co-culture will selectively grow microorganisms that resist these professional phagocytes. An alternative approach for the isolation of novel ARM is also available, which requires the isolation of new amoebal strains by amoebal enrichment on a suitable prey (such as Escherichia coli), and then to search for intra-amoebal microorganisms within the isolated amoebae. Once new potentially pathogenic ARM has been isolated, one should then further assess the potential infectivity of these intracellular microorganisms. The application of macrophages, as an in vitro model to test microbial virulence is also possible.
Further reading:
Detection of Pathogens in Water Using Microarrays
from Timothy M. Straub in Environmental Microbiology: Current Technology and Water Applications
For waterborne pathogen monitoring, regulatory agencies have traditionally focused on developing a single method for an existing or emerging pathogen in water supplies. However, the ability to use a single method to determine all potential pathogens or indicators in a water supply would be particularly advantageous. Such an approach has three major hurdles: 1) sensitive detection of highly dilute pathogens in a water supply, 2) specific detection of pathogens from non-pathogenic near-neighbors, and 3) multiplexed strategies that preserve the sensitivity and specificity of the assay.
Further reading:
For waterborne pathogen monitoring, regulatory agencies have traditionally focused on developing a single method for an existing or emerging pathogen in water supplies. However, the ability to use a single method to determine all potential pathogens or indicators in a water supply would be particularly advantageous. Such an approach has three major hurdles: 1) sensitive detection of highly dilute pathogens in a water supply, 2) specific detection of pathogens from non-pathogenic near-neighbors, and 3) multiplexed strategies that preserve the sensitivity and specificity of the assay.
Further reading:
Low Cost Screening of Multiple Waterborne Pathogens
from Seyrig et al in Environmental Microbiology: Current Technology and Water Applications
A vast array of low cost, simple, rugged, and rapid molecular approaches are emerging for the detection of indicators and pathogens, along with the collection of relevant genotypic information. Loop-mediated isothermal amplification (LAMP) is a relatively new DNA amplification technique, which due to its simplicity, ruggedness, and low cost could provide major advantages to the water industry. In LAMP, the target sequence is amplified at a constant temperature using either two or three sets of primers and a polymerase with high strand displacement activity. Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification. The corresponding release of pyrophosphate results in visible turbidity due to precipitation, which allows easy visualization by the naked eye, especially for larger reaction volumes or via simple detection approaches for smaller volumes. The reaction can be followed in real-time either by measuring the turbidity or the signals from DNA produced via fluorescent dyes that intercalate or directly label the DNA, and in turn can be correlated to the number of copies initially present. Hence, LAMP can also be quantitative. While LAMP is already the method of choice in organizations engaged in combating infectious diseases such as tuberculosis, malaria, and sleeping sickness in developing regions, it has yet to be extensively validated for commonly known waterborne pathogens.
Further reading:
A vast array of low cost, simple, rugged, and rapid molecular approaches are emerging for the detection of indicators and pathogens, along with the collection of relevant genotypic information. Loop-mediated isothermal amplification (LAMP) is a relatively new DNA amplification technique, which due to its simplicity, ruggedness, and low cost could provide major advantages to the water industry. In LAMP, the target sequence is amplified at a constant temperature using either two or three sets of primers and a polymerase with high strand displacement activity. Due to the specific nature of the action of these primers, the amount of DNA produced in LAMP is considerably higher than PCR based amplification. The corresponding release of pyrophosphate results in visible turbidity due to precipitation, which allows easy visualization by the naked eye, especially for larger reaction volumes or via simple detection approaches for smaller volumes. The reaction can be followed in real-time either by measuring the turbidity or the signals from DNA produced via fluorescent dyes that intercalate or directly label the DNA, and in turn can be correlated to the number of copies initially present. Hence, LAMP can also be quantitative. While LAMP is already the method of choice in organizations engaged in combating infectious diseases such as tuberculosis, malaria, and sleeping sickness in developing regions, it has yet to be extensively validated for commonly known waterborne pathogens.
Further reading:
- Environmental Microbiology: Current Technology and Water Applications
- Nanotechnology in Water Treatment Applications
- Metagenomics: Theory, Methods and Applications
- Environmental Molecular Microbiology