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

Area of Science:

• Malariology
• Parasitology
• Molecular Biology

Background:

• Molecular and cellular studies of Plasmodium falciparum rely on limiting dilution cultivation in microplates.
• Slow parasite replication and laborious identification methods hinder these studies.
• A novel high-throughput method for detecting Plasmodium falciparum growth is presented.

Purpose of the Study:

• To develop a high-throughput, non-invasive method for detecting Plasmodium falciparum growth in microplates.
• To facilitate parasite cloning and genetic studies by improving detection efficiency.
• To identify a suitable fluorescent pH indicator for monitoring parasite viability.

Main Methods:

• Surveyed fluorescent pH indicators, identifying 5-(and-6)-carboxy SNARF-1 for its suitable pKa and membrane-impermeant properties.
• Optimized conditions for detecting viable Plasmodium falciparum in 96-well microplates using the dye.
• Applied the method for limiting dilution cloning of genetic cross progeny and transfected parasites.

Main Results:

• 5-(and-6)-carboxy SNARF-1 accurately reported extracellular pH changes associated with parasite growth.
• The dye enabled efficient detection of viable parasites in microplate wells, comparable to manual staining.
• The non-toxic nature of the dye allowed for detection without compromising parasite viability or requiring additional assays.

Conclusions:

• The fluorescence-based assay offers a low-cost, efficient method for detecting viable Plasmodium falciparum in microplate cultures.
• This assay can be readily automated for even higher throughput.
• The method supports advanced genetic studies and parasite cloning.