Sample Processing - Waterborne Pathogens: Detection Methods and Applications - page 86

recovery depends upon many factors, including the secondary concentra-
tion efficiency, operator experience, water quality, etc. whichever technique
is used.
4.4. SECONDARY CONCENTRATION TECHNIQUES
Although after filtration, flocculation, or centrifugation, the sample
volume has been reduced substantially, further concentration is normally
needed. This usually includes a centrifugation step that pellets bacteria, par-
asites, and any debris present in the samples, while viruses remain in the
supernatant. For parasites, depending on water quality and the detection
method used, most often microscopic or PCR-based techniques, additional
purification techniques may be required, e.g. to decrease the amount of
debris and autofluorescing algae that may impair microscopy, or remove
PCR inhibitory compounds.
After centrifugation, viruses remaining in the supernatant can be further
concentrated using ultrafilter centrifugation, PEG-precipition, flocculation,
or ultracentrifugation, which are techniques presented in the following sec-
tions. Here we also present centrifugation, density gradient separation, and
membrane filtration which can all be utilized for the further concentration
of bacteria and parasites. Some discrimination between different pathogen
types is possible at this stage by choice of conditions. However, for specific
enrichment and isolation of a particular pathogen, IMS is required. This is
discussed in Section 4.3.4 . An alternative means of pathogen isolation is the
use of electrical separation techniques, which are discussed in Chapter 6;
such methods are not widely adopted for the manipulation of waterborne
pathogens.
4.4.1. Centrifugation
For pelletting of Cryptosporidum oocyst and Giardia cysts, the ISO
15553:2006 recommends centrifugation at 1100 × g and EPA Method
1623 1500 × g for 15 min, both without braking during deceleration. Sev-
eral other centrifugal forces have been reported, e.g. 3000 × g for 10 min
after flocculation, 61 3300 × g for 30 min, 62 and 4000 × g for 30 min 8 to also
recover bacteria and bacterial endospores after ultrafiltration. Further, Stine
et al. found that centrifugation at 1500 × g followed by staining with Calco-
fluor white, resulted in 52.5% recovery of E. intestinalis spores while increas-
ing the force to 2000 × g decreased the fraction recovered to only 12.8%. 63
However, Dowd et al., detected both E. intestinalis and Cyclospora cayetanesis