A Step-by-Step Guide to Plasmodium Culture Media
If you’ve spent any time in a malaria lab, you know that Plasmodium falciparum is a picky eater.
The goal here is to create a stable, nutrient-rich environment that mimics human physiological conditions while accounting for the common pitfalls of lab life; like the frustrating tendency of buffers to outgas or essential amino acids to degrade.
The media used (RPMI-1640) was developed at Roswell Park Memorial Institute in the 1960s. The "1640" refers to the formulation number assigned during its development, reflecting the extensive empirical (trial-and-error, experiment-based) optimisation that produced the medium.
RPMI-1640 has a fascinating history because it emerged during the period when mammalian cell culture was transitioning from empirical media recipes to more rationally designed formulations. For those like me, wondering what empirical means, it’s approaches that were based on observation, experimentation, and trial-and-error rather than a complete theoretical understanding.
Origin of RPMI-1640
RPMI stands for: Roswell Park Memorial Institute
The medium was developed at the Roswell Park Comprehensive Cancer Center in Buffalo, New York.
The principal developers were:
George E. Moore
Robert E. Gerner
Harold A. Franklin
during the 1960s.
The general components and amounts in 1L RPMI are:
RPMI 1640: 10.44 g (The nutritional backbone, containing l-glutamine at a final conc. of 2mM). This is 5.22g if only making 500 mL
HEPES: 5.96 g (Your primary buffering agent). If making 500 mL, add 12.5 mL of 1M HEPES. If you buy the powdered RPMI, it likely comes with this already.
NaHCO3: 58 mL of 3.6% (final is 2g/L ; For pH stability and gas exchange). Sodium bicarbonate is notorious for outgassing (releasing CO2), which can cause your pH to drift upward over time. To combat this, we add it just before use.
Hypoxanthine: 50 mg (200uM, Essential for parasite purine salvage)
Gentamicin: 20 ug/mL stock (Your antibiotic shield). For 1L you can add 20 mg of gentamicin
Ultrapure/sterile H2O: 960 mL
1M NaOH: For dissolving the hypoxanthine
Conc. HCL and NaOH: For final pH adjustment
REFERENCES:
1. Lopez-Perez, M., & Seidu, Z. (2022). Establishing and Maintaining In Vitro Cultures of Asexual Blood Stages of Plasmodium falciparum. Methods in Molecular Biology.
2. Maier, A. G., & Rug, M. (2013). In vitro culturing Plasmodium falciparum erythrocytic stages. Methods in Molecular Biology.
3. Trager, W., & Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science, 193(4254), 673–675.
4. World Health Organization. (2023). World Malaria Report 2023. Geneva: WHO.
