A highly efficient in vitro cloning procedure for asexual erythrocytic forms of the human malaria parasite Plasmodium falciparum.

Abstract

A very reliable and productive technique for cloning of Plasmodium falciparum in vitro is proposed, as demonstrated by successive limiting dilution of suspensions of asexual erythrocytic forms of the NF 54 strain. The introduction and the study of reliable clones is of extreme importance for a better understanding of the behaviour of the parasite, also in field conditions. The method is rapid, simple and efficient. The growth of the clones was individually monitored and the culture conditions were constantly adjusted during their stay in recipients of increasing size. A yield of 18/96 (18.75%) of provisional clones was obtained, while the supercloning phase resulted in 16/80 (20%) positive cultures. The probability that the latter were derived from a single progenitor is very high (99%). It was shown that three randomly selected clones (A1A9, A1B11, and A1C10) have excellent growth characteristics before and after cryopreservation, and after a longer period of culture in standard conditions.