Sample Processing - Waterborne Pathogens: Detection Methods and Applications - page 71

all treatments reduced viral adsorption to the filter as compared to unblocked
filters with the overall highest recovery for all three viruses for 1% FBS. FBS
is a commonly used blocking agent but accidental contamination during
blocking may result in microbial growth during storage and transport in
the presence of FBS. Therefore, Hill et al. investigated the use of NaPP as
an alternative blocking agent for the recovery of various vegetative bacteria,
bacterial endospores, viruses, and Cryptosporidium oocysts, and no statistically
significant difference was observed between filters coated with 5% FBS and
0.1% NaPP when the water and back-flush solution was amended with 0.1%
NaPP. 21 Decreasing the NaPP concentration to 0.01% gave recoveries that
were not significantly different from those with 0.1%. In the same study, it
was also shown that addition of 0.01% Tween80 and Tween20 to the back-
flush solution increased microbial recovery while addition of Tween80 to
the water sample increased fouling of the filter which decreased the perme-
ate flow, thus resulting in longer filtration times. Whenever Tween was used,
0.001% Antifoam A was added to prevent foaming. Worth noting is that it
also was shown that no inhibition of PCR, or RT-PCR, was observed when
a solution of 0.01% NaPP, 0.1% NaPP, or 0.01% Tween80 was seeded with
two different viruses, suggesting that the additives would not impair molecu-
lar detection.
Polaczyk et al. investigated the effect of seed level and NaPP in the
water, and compared direct collection of the retentate with elution and
back-flushing, respectively, for recovery of vegetative bacteria, endospores,
bacteriophages, Cryptosporidium oocysts, and microspheres. 8 Seeding with
100 and 1000 cfu or pfu per organism or particle gave very similar results
for all organisms. Addition of 0.01% NaPP to the water increased the mean
recovery for all organisms from 46% to 71%, but a significant difference was
seen only for Bacillus atrophaeus endospores. NaPP did not decrease recovery
for any single microorganism. The difference in average mean recovery for
all organisms between back-flushing and elution was small and not signifi-
cant, but back-flush increased recoveries of MS2-phages, Salmonella enterica ,
B. atrophaeus endospores, and Cryptosporidium parvum oocysts. However, in
some cases elution decreased recovery compared to direct collection of the
retentate, while back-flushing always resulted in an increase in recovery.
Comparisons of recovery rates between studies are very difficult since
so many parameters differ. Filters (brands and sizes), water quality, seed-
ing organisms (levels and detection methods), blocking conditions, filtration
method and samples amendments, elution techniques and buffer composi-
tion, can be combined in an endless number of ways. Some examples of