Effect of oil treatment on progeny virus production by PR8 as measured by Fluorescent focus assay (FFA). After MDCK cells in 24-well plates were infected with oil-treated and untreated virus for 48 h, five microliters of supernatants were removed, serially diluted and added to confluent MDCK cells in 96-well plates. After incubation for 7 h, IAV nucleoprotein (NP) was detected using an Alexa Fluor 488 (green) labeled antibody. Panels: (A) MDCK cells unexposed to virus, but stained with anti-NP antibody. Panels (B-F) MDCK cells exposed to PR8 treated with different dilutions of essential oil: (B) 1:1,000 (C) 1:2,000 (D) 1:3,000 (E) 1:4,000 (F) 1:6,000, (G) untreated PR8, (H) PR8 treated with control oil at a 1:1,000 dilution. Panels I-L were fluorescence images merged with corresponding brightfield images to show MDCK cell morphology: (I) PR8 treated with essential oil 1:4,000, (J) PR8 treated with essential oil 1:6,000, (K) untreated PR8, (L) PR8 treated with control oil 1:1,000 (merge). Bottom panel: Infectivity as reflected by the percentage of cells in which IAV NP was detected. The results represent the mean ± SEM from three independent experiments.