Evaluation and molecular characterization of human adenovirus in drinking water supplies: viral integrity and viability assays

Fongaro, Gislaine; Nascimento, Mariana A do; Rigotto, Caroline; Ritterbusch, Giseli; da Silva, Alessandra D’ A; Esteves, Paulo A; Barardi, Célia R M

To infer the presence of infectious HAdV particles, water samples were previously treated with 10 U/mL penicillin, 10 μg/mL streptomycin and 0.025 μg/mL amphotericin B. The treated samples were inoculated (0.25 mL) at a non-cytotoxic dilution, in triplicate, in A549 cells for the plaque assay, as described by Cromeans et al. (2008) [43] and Rigotto et al. (2011) [44] . The cells were incubated for 1 h at 37°C with rotation every 15 min. The cells were then removed and overlaid with 0.6% Bacto-agar (previously melted) in high glucose DMEM 2X (high-glucose Dulbecco's Modified Eagle's Medium) containing 4% FBS, 0.1 mM sodium pyruvate, 10 U/mL penicillin, 10 μg/mL streptomycin and 26 mM MgCl 2 . The samples were then incubated at 37°C for 7 days. Then, the agar overlay was removed, and the cells were stained with 20% Gram's crystal violet and the plaques counted macroscopically.