Existing Methods of Waterborne Pathogen Detection - Waterborne Pathogens: Detection Methods and Applications - page 58

Figure 3.7 Overview of the USEPA 1623 method for the detection of Cryptosporidium .
To clarify in step 3 a minimum of 2 washes of 600 mL each should be performed. With
thanks to Scottish water for provision of the images. Source: Adapted from Ref. 32 . (For
color version of this igure, the reader is referred to the online version of this topic.)
Each stage of the monitoring process reduces the initial 1000L to
smaller and smaller volumes, eventually reaching the 50-100µL for the
microscope slide. Throughout the process there are significant losses of
(oo)cysts with acceptable recovery rates for this method at just over 10%.
Water companies in the UK report around 30% recovery as an average. It
is clear from the above discussion that sample processing is key for effec-
tive monitoring of waterborne protozoan. It is extremely unlikely that any
detection technology could reliably detect to the single (oo)cyst level in
1000 L. This topic is discussed further in Chapter 4.
Drawbacks of this approach are the time required for detection, the poten-
tially low recovery rates, the need of expensive fluorescent reagents, as well as
the requirement for highly trained technicians and lab equipment (especially
the microscope). The protocol can take up to 3 days and no indication of spe-
cies or viability is given. Molecular methods have been developed for these
pathogens, though there has not yet been agreement over what the most
appropriate nucleic acid targets are, or whether it is possible to determine
viability simultaneously. Additionally, reliable single (oo)cyst detection is not
yet available. Developments in molecular methods are covered in Chapter 8.
Cyclospora can be recovered with the methods used for Cryptosporidium .
However, no vital dye assay is currently available, so samples require a 2 week
incubation period to check for sporulation to determine viability. 31 Acid fast
staining techniques, for the diagnosis of the organism, are also problematic
and not recommended for detection of Cyclospora in environmental samples.