flashBAC Manual - Oxford Expression Technologies

Remove the liquid overlay from the dishes and replacewith 1 ml diluted Neutral Red stain (see list at the start).Incubate for 3-4 hours at 28°C.Tip off the stain (into disinfectant) and invert dishes (placeon tissue paper which can then be discarded byautoclaving). Replace lids.Leave the dishes in the dark, in the inverted position, forthe plaques to clear. This may take a few hours or mayoccur very rapidly, depending on the strength of theNeutral Red.• LacZ-positive virus plaques (e.g. virus produced using thecontrol transfer vector) can be stained using X-gal ratherthan Neutral Red. Add 1 ml of appropriate insect cellculture medium containing 15 µl (2%w/v) X-gal (in DMF)and incubate for at least 5 hours (may need overnight) at28°C. Plaques will appear blue in colour.12 After staining, select one set of duplicate dishes withbetween 10 and 30 plaques (ideally) and count them.Calculate the average number of plaques for that dilution.To determine the virus titre use the following calculation:Titre of virus (pfu/ml) = average plaque count xdilution factor* x 10***multiply by the inverse of the dilution used to count theplaques** multiply by 10 because only 0.1 ml was applied to eachdish. Example: 25 plaques (average) on the 10 -6 dilutionOXFORD EXPRESSION TECHNOLOGIES 36