from Theron et al. in Nanotechnology in Water Treatment Applications

Flow cytometry (FC) detects and quantify light scattering from fluorescent-labeled cells that have crossed a laser beam. A single sample can be analysed within 3-5 min with a quantification limit of approximately 200 cells/ml. FC, although an optical detection method, is used in combination with molecular techniques. Bacterial cells in water have been monitored with flow cytometry through nucleic acid staining or targeting specific cells with antibodies or FISH hybridization.

FC is a valuable tool to differentiate between viable, intermediate and nonviable cells. Baclightbacterial viability kit (Live/Dead kit), widely used in flow cytometry, double stains nucleic acid with SYTO dyes (green fluorescence) and propidium iodide (PI) (red fluorescence). SYTO dyes stain the nucleic acid of all the cells, resulting in green fluorescence. The cells are afterwards stained with PI which can only move into membrane compromised cells, staining the nucleic acid and resulting in red fluorescence. The disadvantage is that cells can be dead without showing membrane damage and hence is this rather an assay representing membrane damage than cell viability. Calculating the nucleic acid content has also been used as an indicator of cell viability. The theory is that cells with higher cell viability reproduces at a higher rate and therefore will contain more copies of their genome. Care must be taken with the interpretation of results obtained from this approach. Bouvier et al. investigated the varied correlation between different nucleic acid contents and metabolic activities of subpopulations from a wide range of environmental communities.

FC combined with nucleic acid staining enable researchers to investigate the growth potential of microbial pathogens in natural waters. Vibrio cholerae, the causative agent of cholera, was shown through FC and SYBR Green nucleic acid staining to grow in different freshwater samples. This contradicted previous opinions that natural waters do not have sufficient nutrients to support the growth of this pathogen. Combining these experiments with assimilable organic carbon (AOC) concentrations it was concluded that V. cholerae would proliferate in water with a minimum AOC of 60 mg/l. The same research group investigated the growth potential of E. coli O157 in freshwater samples using the same methodology. E. coli O157 was able to grow in freshwater samples with low carbon concentrations, once again contradicting previous opinions.

Fluorescence activated cell sorting (FACS) makes FC even more indispensible for detecting and differentiating between microbial pathogens in water. Cells with specific nucleic acid targets can be labeled with FISH probes, quantified and separated with FC-FACS. These cells can then be subjected for further genetic and biochemical analysis. Catalyzed fluorescent reporter disposition-FISH and molecular beacons are now incorporated into FC-FACS to increase stain sensitivity and overcome the problem of sorting cells present in low numbers. FC-FACS-FISH has also been applied to sequence previously unsequenced microorganisms and cultured previously uncultured microorganisms from environmental samples.

Tags: Microbial Detection | Pathogen Detection | Biodiagnostics | Biodetection Assays | Biomolecular Detection