Cultivation of the Parasites 485 



the deeper part of the corpuscular layer, thus escaping the leukocytes at the top, 

 and planted at the bottom of each tube of serum. It is thought to be advan- 

 tageous to use culture tubes with flat bottoms. A still better method is the 

 introduction of a paper disk into a half-inch tube, abouthalf an inch below the 

 surface of the serum, and then place one- or two-tenths of a cubic centimeter 

 of corpuscles upon it. Under these circumstances all of the plasmodia are 

 said to grow and segment. _ Two or three generations of parasites grow in such 

 cultures, then the plasmodia begin to die out, so that if the culture is to be 

 perpetuated, they must be transplanted to freshly prepared blood-corpuscle 

 tubes of the same kind. The method of transplantation recommended is so 

 very simple: a drop of the culture is drawn into a iine (not capillary) glass 

 pipette and then followed by about five times the volume of the fresh corpuscle 

 suspension. These are mixed in the pipette, care being taken not to mix air 

 with the blood, and are then transferred to the new media in the same manner 

 as in making the original inocul ation. The transplantation should be done within 

 five hours of the time of maximum segmentation, and therefore every forty- 

 eight hours for the tertian and aestivo-autumnal parasites. All species of the 

 Plasmodia have been successfully cultivated by these means. The parasites 

 have also been grown in red blood-cells in Lock's solution, free of calcium chlorid 

 and in the presence of ascitic fluid. 



According to Bass and Johns, the parasites grow in the corpuscles, not upon 

 them as believed by Koch. They are destroyed in a few minutes in vitro by 

 normal human serum or by all the modifications of it that they have tested. 

 This fact, together with numerous observations of parasites in all stages of de- 

 velopment apparently within the corpuscles render untenable the idea of extra- 

 corpuscular development. Leukocytes phagocytize and destroy malarial para- 

 sites growing in vitro only when they escape from their red-corpuscle capsule or 

 when the latter is perforated or becomes permeable. 



The substance of the malarial plasmodium is very different in consistency from 

 that of the blood-cells, and therefore they cannot pass through the smallest 

 capillaries like the more yielding fluid-like red blood-cells. That the consistency 

 of the protoplasm of the parasite is less yielding than that of the red blood-cell 

 is shown by the fact that when a small quantity of a culture containing large 

 parasites is spread over a slide with the end of another slide, the parasites are 

 dragged to the end of the spread though the red blood-cells are left behind. 

 Large asstivo-autumnal plasmodia are round or oval; the tertian variety are 

 more or less flattened. As a result of their unyielding consistency, malarial 

 parasites lodge in the capillaries of the body, especially where the current is 

 weakest, and remain and segment. In the meantime other red corpuscles are 

 forced against them and if in a favorable situation, one or more merozoites pass 

 directly into the other cells. When the segmented parasite has become sufli- 

 ciently broken up it can pass through the capillary into the circulating blood where 

 the remaining merozoites are almost instantly destroyed. 



They further observed that calcium salts added to cultures of sestivo-autumnal 

 parasites caused hemolysis of the infected, possibly also of non-infected red 

 blood-cells. Such salts have no effect on the corpuscles of normal blood, pos- 

 sibly because of the precipitation of other substances from the serum. The 

 amount of calcium necessary to cause hemolysis of malarial blood is only slightly 

 in excess of the quantity present in normal blood and possibly might be reached 

 by the ingestion of considerable quantities of calcium in drinking water or 

 food. They speculate that malarial hemoglobinuria may be the result of the 

 presence of an excess of calcium in drinking water. 



Bass and Johns believe that quinine has no direct effect upon the malarial 

 parasites, but effects its curative influence by rendering the substance of the 

 corpuscles more permeable to the all-sufficient destructive influence of the 

 serum. The quinine would then affect only the parasites in the circulation, and 

 not those lodged in the capillaries, which would not be reached until they had 

 segmented. The effect of quinine is said to be defeated by influences such as 

 diet, exertion, etc., which increase the dextrose content of the blood, whereby 

 the permeability of the red blood-cells seems to be decreased. It is hopedthat a 

 better understanding of the principles involved in the treatment of malaria may 

 result from the study of the organism in culture by which empiricism may be 

 exchanged for rationalism. 



