Studies on Malarial Parasites Vii. Methods and Techniques Por Cultivation* by Quentin

When Laveran (1) discovered living malarial parasites over 65 years ago in the blood of patients with certain fevers, a new era in the study of malaria was initiated. This discovery of malarial organisms and the subsequent experimental proof by a series of workers that they were the etiological agents of the disease failed, however, to lead to the immediate successful cultivation of the parasites. The cultural methods being developed at that time for bacteria were tried in efforts to grow these erythrocytic parasites using a variety of materials, ranging from marsh mud to human blood. Claims were made for successful cultivation but they remained unconfirmed when the work was repeated. Sacharoff, 1890, and Rosenbach, 1890 (see Thayer and Hewetson, 2) tried the use of leeches to get development and multiplication outside the human body, but these workers merely got survival and only partial development of the parasites. The search for methods of cultivation continued after Ross and the Italian workers at the turn of the century discovered the mosquito cycle and transmission of the malarial parasite. Little success was obtained, however, until Bass and Johns (3) reported cultivation of two species of malarial parasites. The original method consisted of the addition of glucose to 2 cc. of defibrinated blood, incubation of the mixture, and examination at intervals for the parasites which were supposed to grow on the top layer of the sedimented red blood cells. This method was widely used and modified in some cases to study the development of the plasmodia and even to diagnose malaria in India. Claims were made for the development of three generations of human plasmodia by this method (4, S), but quantitative figures were not given and it is extremely difi5cult to evaluate these claims. During the subsequent years, a variety of methods was tried with avian malarial parasites, but little success was obtained (reviewed by Trager, 6). The best results were obtained by Trager (6,7) working with the avian parasite, Plasmodium lophurae. He studied the conditions affecting the survival of this parasite in v&o and was able to obtain survival up to 16 days at 40-41”C. The medium contained a red cell extract in balanced salt solution, glutathione, glucose, serum, embryo extract, and calcium pan-