Optical Detection Technologies for Waterborne Pathogens - Waterborne Pathogens: Detection Methods and Applications - page 120

(fast, 10,000 cells s −1 but potentially risky with aerosol forming pathogens)
approach to isolate target populations for further analysis or directly for
detection.
In comparison with other methods, flow cytometry often delivers an
undercount compared to microscopy because sometimes clusters could be
perceived by a flow cytometer as one cell, although it avoids false negatives
in microscopy due to viewer fatigue. 21 Compared to culture-based meth-
ods, flow cytometry often reports an overcount as 1 cfu could form from
more than one initial cell. 20
Advantages of flow cytometry are that it is faster and more automated
than culture-based techniques and that microorganisms are undisturbed by
the analysis and therefore remain available for further testing, if required.
It is also compatible with many different fluorescent detection approaches,
allowing for a range of information to be collected ( Table 5.1 ).
Some of the disadvantages of flow cytometry are that considerable
processing is still needed and, especially for those microorganisms with
low detection limits, flow cytometry may still have to be combined with
microscopy. Flow cytometry is also challenged by the complex matrices of
environmental samples which may contain significant volumes of particu-
late matter of a similar size, some of which might autofluoresce, preventing
the use of certain fluorophores.
In terms of sampling considerations, for samples to be analyzed by flow
cytometry the biggest challenge is to avoid aggregation of particles as they
can cause blockage of the cytometer and also lead to undercounts. The
use of resuspension in tetrasodium pyrophosphate has been suggested to
reduce aggregation. 20 Immunomagnetic separation is another useful option
to reduce potential blockages ( Figs 5.6 and 5.7 ).

Table 5.1 Molecular methods for microbial sensing compatible with flow cytometry
Assay type Criteria Specific examples

Nonspecific Nucleic acid stains Picogreen, SYBR-Green

Cytoplasm stains Rhodamine

Specific Antigen-based Immunoassays, fatty acid signatures

Nucleic acid-based DNA/RNA probes, FISH PCR,

sequencing, ribotyping

Cell functioning Membrane integrity Propidium iodide

Enzyme activity Tetrazolium salts, fluorogenic

enzyme assays, esterase assays

Membrane activity Rhodamine, Bis-oxonol

Adapted from Table 2 in Ref. 22 .