2.3. Virus recovery efficiency
AS samples, collected from Tudhoe Mill WWTP, were seeded
with the dsDNA coliphage T4 (NCIMB, UK) and left for 15 min
prior to fixing. Triplicate samples, with and without the
seeded T4 coliphage, were then prepared and analysed
following both the optimised protocol and that of Brussaard
et al. (2010). The seeded T4 abundance was determined by
FCM (0.91 ± 0.04 109) and, for comparison, by plaque assay
(1.0 ± 0.17 109). Briefly 20 mL of the host isolate Escherichia coli
was suspended in 8 mL of sterile sloppy agar (0.5% agar in
nutrient broth medium) together with 20 mL of filter-fertilised
(0.2 mm) T4 coliphage culture. The sloppy agar was then
poured over a pre-warmed (37 C) nutrient agar plate and
incubated for 2 days at 37 C. Plates were checked after 24 and
48 h for plaque formation. The FCM seeded concentration of
0.91 109 was used for calculations