Toluidine Blue Staining of Coccidia in Cultured Animal Cells

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Abstract
Cultured animal cells infected with various species of Eimeria (coccidia) from chickens are washed in Hanks balanced salt solution (HBSS) and fixed in 5% formalin in HBSS. The fixed cultures are washed briefly in distilled water to remove HBSS salts and then dehydrated in a series of mixtures of 40 to 50% ethanol with increasing concentrations of tertiary butanol (TB) and decreasing concentrations of distilled water. Cultures are placed for 1 min in a mixture of 2 parts ethanol :TB (25:75) and 1 part 0.05% toluidine blue O in McIlvaine buffer (pH 6.0), followed by 1 min in 0.05% toluidine blue O in McIlvaine buffer (pH 6.0). The stained cultures are dipped for 1–2 sec in TB, allowed to dry and mounted permanently on slides. Cover-slip cultures fixed and stained by this procedure 8 years ago have not faded or discolored. The alcohol mixtures, formalin in HBSS, stain and buffer can be prepared in large volumes and stored indefinitely. The staining procedure has proven to be rapid and dependable with a variety of cell types in monolayer cultures in research and teaching applications.