A high-throughput method to detect Plasmodium falciparum clones in limiting dilution microplates

BACKGROUND: Molecular and cellular studies of Plasmodium falciparum require cloning of parasites by limiting dilution cultivation, typically performed in microplates. The parasite's slow replication rate combined with laborious methods for identification of positive wells has limited these studies. A new high-throughput method for detecting growth without compromising parasite viability is reported. METHODS: In vitro parasite cultivation is associated with extracellular acidification. A survey of fluorescent pH indicators identified 5-(and-6)-carboxy SNARF-1 as a membrane-impermeant dye with a suitable pK(a )value. Conditions for facile detection of viable parasites in 96-well microplates were optimized and used for limiting dilution cloning of genetic cross progeny and transfected parasites. RESULTS: 5-(and-6)-carboxy SNARF-1 is a two-emission wavelength dye that accurately reported extracellular pH in parasite cultures. It readily detected parasite growth in microplate wells and yielded results comparable to labour-intensive examination of Giemsa-stained smears. The dye is non-toxic, allowing parasite detection without transfer of culture material to additional plates for separate assays. This dye was used with high-throughput limiting dilution culture to generate additional progeny clones from the HB3 × Dd2 genetic cross. CONCLUSIONS: This fluorescence-based assay represents a low-cost, efficient method for detection of viable parasites in microplate wells; it can be easily expanded by automation.