title: Pittsburgh viral preparation from raw sewage
organism: metagenomes
description: 5L of untreated wastewater was obtained from the wastewater treatment plant in Pittsburgh, Pennsylvania (USA) in December 2009 and stored for 2 hours at 4*C before processing. Virions were concentrated from the wastewater sample by organic flocculation based on the procedure previously described (Calgua et al., J. Virol Methods, 2008, vol 153, p.79-83). Briefly, 100ml of pre-flocculated skim milk solution (pH 3.5) was added to 10 L acidified raw sewage (pH 3.5) and mixed for 8 hours. Flocculants were allowed to settle and then centrifuged. The flocculated viral concentrate was resuspended in 15 ml phosphate buffer and then eluted in 30 mL of 0.25 M glycine pH 9.5 for 45 min at 4* C by slow agitation with vortexing. Suspended solids were separated by low speed centrifugation at 7,500 g for 30 min at 4* C and the high pH of the supernatant was stabilized by adding 20 mL of 2X PBS. Virions present in the supernatant were concentrated by ultracentrifugation at 100,000 g for 1 hr at 4* C and resuspended in phosphate buffer (1:2 v/v of 0.2 M Na2HPO4 and 0.2 M NaH2PO4).