Blastocystis hominis: A simplified, high-efficiency method for clonal growth on solid agar

Colony growth of protozoan parasites in agar can be useful for axenization, cloning, and viability studies. This is usually achieved with the pour plate method, for which the parasite colonies are situated within the agar. This technique has been described for Giardia intestinalis, Trichomonas vaginalis, and Entamoeba and Blastocystis species. Extracting such colonies can be laborious. It would be especially useful if parasites could be grown on agar as colonies. These colonies, being exposed on the agar surface, could be conveniently isolated for further investigation. In this study, we report the successful culture of B. hominis cells as colonies on solid agar. Colonies were enumerated and the efficiency of plating was determined. It was observed that B. hominis could be easily cultured on agar as clones. The colonies were dome-shaped and mucoid and could grow to 3 mm in diameter. Flow cytometric analyses revealed that parasite colonies remained viable for up to 2 weeks. Viable colonies were conveniently expanded in liquid or solid media. Scanning electron microscopy revealed that each colony consists of two regions; a dome-shaped, central core region and a flattened, peripheral region. Older colonies possessed numerous strand-like surface coat projections. This study provides the first report of clonal growth of B. hominis on agar and a simple, effective method for cloning and expansion of B. hominis cells.