title: Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples.
abstract: The influence of inoculum size and cell culture density on virus titer by cytopathic effect or plaque assay was studied using poliovirus type 1 and BGM (Buffalo green monkey) cells as a model for this evaluation. With a plaque assay system, a linear relationship was observed for an inoculum size of up 1 mL/25 cm2; a marked decrease in the number of plaques was observed when over 1 mL of sample was inoculated on this surface area. Cell culture density also affected virus titer; maximal titers were observed when cells were seeded at 25 000 to 75 000 cells/mL and incubated for 6 days before infection with the virus. Viral density, evaluated as most-probable-number and measured by cytopathic effect under liquid overlay, revealed that the viral titer was similar up to 1 mL inoculum and increased only when over 1 mL was inoculated. Cell density had no significant effect on the viral titer measured by the most-probable-number method and cytopathic effect. Inactivation of inoculum due to an incubation temperature of 37 degrees C for a short period was shown to be minimal for poliovirus type 1, reovirus type 2, coxsackievirus B-5, and the simian rotavirus SA-11. Longer inactivation time led to a 2 logs reduction of the infectious titer of coxsackievirus B-5 (in 48 h) while the other viruses showed a significant reduction in titer only after 96 h.
mesh_terms: D000818:Animals; D002452:Cell Count; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D029822:Enterovirus B, Human / Q000254:growth & development; D004783:Environmental Microbiology; D017955:Poliovirus / Q000254:growth & development*; D012087:Reoviridae / Q000254:growth & development; D012190:Retroviridae / Q000254:growth & development; D013696:Temperature; D010948:Viral Plaque Assay; D014780:Viruses / Q000254:growth & development*