Toxoplasma gondii plaque assays revisited: Improvements for ultrastructural and quantitative evaluation of lytic parasite growth.

Lytic growth of intracellular Toxoplasma gondii tachyzoite stages over a period of days results in plaques within mononolayers of host cells. Plaque assays are in frequent use to isolate single clones and to investigate invasion, replication and egress over a longer time frame. To allow correlating plaque morphology and/or size with ultrastructural examination of individual parasites we introduce a simple protocol for correlative light and electron microscopy (CLEM) of entire plaques. We also illustrate the advantages of visualizing only the boundaries of plaques by staining for infected cells ('positive staining') rather than the traditional staining of the intact cell monolayer, thus outlining the area of lysed cells ('negative staining'). Tachyzoites expressing β-galactosidase of Escherichia coli are an easy to visualize histochemical marker for this purpose. Quantitative measurements of plaque area with our compiled user-friendly ImageJ macros are compared to commercial software for ease and shown to be more accurate for some applications. Finally, a chemically defined medium is shown to be superior to the fetal bovine serum-containing medium for plaque assays, resulting in larger plaques. The reported additions and changes of the plaque assay procedure offer improved ways to analyze subtle differences in invasion, pathogen growth and egress. Our chemically defined medium will improve standardization of e.g. drug screening assays.