Use of Various Buffers for In vitro Cultivation of Malarial Parasites

Abstract

Three organic sulfonic acids, BES, TES, and HEPES, were shown to be better buffers than glycylglycine for in vitro cultivation of Plasmodium knowlesi and P. falciparum. Over a concentration range from 10 to 80 mM, in doubling multiples, BES at 40 mM gave the best growth and pli maintenance for P. knowlesi and TES at 40 mM gave the best results for P. falciparum. With TES in the culture medium there was invasion of a significant number of new host red cells and a definite multiplication of P. falciparum after 48 hr. In the presence of TES, P. falciparum at high parasitemias (20 to 50%) has been cultured for 20 to 22 hr in order to obtain large quantities of older stages of the parasite for biochemical studies. Previous experiments on in vitro cultivation of Plasmodium knowlesi demonstrated that the parasitized erythrocytes metabolize large quantities of glucose to lactic acid as compared with normal erythrocytes. With the utilization of glucose, there is a shift in pH caused by the production of lactic acid. The pH of the culture was maintained in the physiological range up to 12 hr but then it started to decrease below required physiological levels for maintenance of cellular integrity. A search for other buffers to counteract the deleterious effects of lowered pH during culture resulted in the finding that glycylglycine when added to the medium would increase the buffering capacity without detectable toxicity to the parasites (Geiman et al., 1966). Since 1966, we have included, as a standard procedure, glycylglycine as a supplementary buffer for in vitro cultivation of P. knowlesi and P. falciparum. The buffering capacity of glycylglycine has not been entirely satisfactory for in vitro cultivation of P. falciparum, where 48 hr of incubation is required for the completion of one developmental cycle. Figure 1 illustrates the changes in pH that occur during the in vitro cultivation of P. falciparum. The initial pH of the medium was adjusted to 7.45. When the blood was added to the medium, there was a Received for publication 16 February 1973. * This work was supported by the Research and Development Command of the U. S. Army (DADA 17-70-C-0120) and from the NIAID, U. S. Public Health Service (AIO-9558-01). This is paper number 1099 from the Army Research Program on Malaria. slight temporary rise to 7.5, undoubtedly due to the loss of CO2 during the handling of the blood. After the initial fluctuations the pH decreased gradually to about 7.36 over the first 12 hr of the culture period. The decreases in pH accelerated after that point so that at 31 hr the pH was 7.01. From 31 hr until termination of the experiment, the decrease slowed somewhat. The pH at 49 hr was 6.86. This experiment indicates that the search for other buffers to counteract the deleterious effects of lowered pH is desirable. Recently Eagle (1971) tested 16 different organic compounds for their buffering capacity in a mammalian cell culture system. Out of these, three compounds, N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES, pKa = 7.15), N-tris (hydroxymethyl) methyl-2aminoethanesulfonic acid (TES, pK, = 7.50), and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES, pKa = 7.55) were selected for experiments designed to improve the buffering capacity of our culture system for in vitro cultivation of P. knowlesi and P. falcip-