Sample Processing - Waterborne Pathogens: Detection Methods and Applications - page 85

pore size of ultrafilters will increase the amount of debris in the con-
centrated material. This will affect the size of the pellet which, in turn,
affects the IMS step in the protocols for Cryptosporidium and Giardia
detection as several separate IMS reactions may have to be performed. It
will also increase the background of fluorescent material in the micro-
scopic analysis and make the correct identification of oocyst and cysts
more difficult. If molecular detection is used, it is likely to increase the
amount of PCR inhibitory substances for example humic acids will be
higher if ultrafiltration is used. The fraction of target DNA in the total
DNA preparation will decrease if no further purification, such as IMS,
is performed.
4.3.6. Continuous flow centrifugation
Centrifugation as a technique is discussed further under the secondary con-
centration techniques, as it normally processes a smaller volume of solu-
tion after one of the above-described primary techniques. However, the
development of continuous flow centrifugation allows for the processing
of larger volumes.
In 2002, Borchard and Spencer presented a continuous separation chan-
nel centrifugation, adapting existing blood cell separators for the purpose
of waterborne pathogen concentration. 58 The advantages of this approach
were the simultaneous concentration of many pathogens, suitability for a
wide range of matrices, and high reproducibility. The main disadvantage was
related to the achievable flow rate, with the authors finding maximums of a
few 100 mm min −1 depending upon the design. This method was applied to
Cryptosporidium , Giardia , and E. coli .
In 2006 Zuckerman and Tzipori developed a portable continuous flow
centrifuge system capable of simultaneous recovery of C. parvum , Giardia
intestinalis , and Encephalitozoon intestinalis , with average recovery rates of
67%, 47%, and 72%, respectively. 59 However, there was a high degree of
variability in the reported recovery rates. The authors also tested 1000L
of tap water spiked C. parvum achieving a 35% average recovery and 10 L
oocyst spiked source water samples, reporting 58% recovery. Zuckerman
and Tzipori conclude that these recovery rates exceed what is acceptable
in Method 1623, and compare favorably to filter results. As mentioned, we
saw that manufacturers claim over 70% recovery rates for their filter systems.
However, evidence suggests recovery rates do not always reach these levels, 60
as confimed by communication with water utilities (personal communi-
cation). This variation in recovery rate is observed for all methods as the