Fluorescent Focus Assay of Viruses on Cell Monolayers in Plastic Petri Plates

Publisher Website
Google Scholar
Abstract
A fluorescent focus (FF) assay technique for quantitating viruses is described. Plastic petri dishes are used without coverslips, thus enabling direct examination of fluorescing infected cells attached to the dish. Benefits derived from using this procedure include minimal mechanical manipulations (thus saving time and minimizing damage of coverslips), large surface areas for examination, high precision and high sensitivity capabilities. Human amnion (FL) cells were used for this study because of their extremely low background fluorescence, high susceptibility to several viruses and their tenacious adherence to the dishes. Four-8x105 cells were used per dish, each of which possessed a surface area of about 800 mm2. Various dilutions of viruses (AAV, (adeno-associated viruses) adenoviruses and herpes virus) were inoculated, the dishes incubated for appropriate periods of time, cells rinsed, fixed and stained in the dish using the indirect Coombs technique. FF were counted at low magnifications so as to view the largest field practical for easy recognition. Dose-response experiments indicated that the method is a valid quantitative procedure. Multiple FF titrations showed a standard deviation of about 8%.