STANDARD ^METHODS AND REAGENTS 473 



enriched by the addition of an equal volume of sterile rabbit's serum for 

 primary cultures, or of sterile defibrinated blood for subculturing. The mix- 

 ture is tubed in three cubic centimeter quantities and is inactivated by 

 heating at 56° C. for thirty minutes. It is then incubated to test for sterility. 



Malarial parasites and piroplasms. The methods used for the 

 cultivation of these two groups of organisms is the same. The 

 procedure consists in growing the parasites in a biological fluid — 

 serum, ascitic or hydrocele fluid — plus blood cells and a small 

 amount of dextrose. The earlier investigators believed that anaero- 

 bic conditions were essential and that the removal of leucocytes by 

 centrifugation greatly reduced the mortality of the extracellular 

 forms, but subsequent work tends to indicate that neither of these 

 precautions is necessary. The method adapted from the work 

 of Bass and Johns (1911, 1912, 1914), is as follows: 



Primary culture. Blood containing recently segmented or, in the case of 

 the piroplasms, budded forms is drawn into a container in which there is a 

 sufficient quantity of a fifty per cent solution of dextrose so that there will 

 be 0.1 of a cubic centimeter of the solution to each ten cubic centimeters of 

 blood. The mixture is defibrinated by stirring and is distributed under 

 aseptic conditions into culture tubes in from five to eight cubic centimeter 

 amounts. The tubes are incubated in the vertical position at 38° -40° C. Cells 

 from the upper layers of the column are examined for parasites. 



Sub-cultures. It is not easy to carry cultures of these parasites through 

 many asexual generations. Bass and Johns obtained growth following trans- 

 fer of the primary culture into tubes containing the serum and erythrocytes 

 of normal defibrinated blood plus dextrose. They obtained these components 

 by centrifuging normal defibrinated dextrose-blood until the leucocytes were 

 separated from the red blood cells and the serum into the "buffy coat." The 

 serum was pipetted into culture tubes without disturbing the layer of leuco- 

 cytes, and the red blood cells were removed by passing a pipette through 

 the layer of white cells and drawing off the erythrocytes. The primary cul- 

 ture is centrifuged in the same manner, and infected red blood cells free 

 from leucocytes are used to seed the sub-culture. Subcultures rarely last 

 over three generations, and the highest recorded number of generations in 

 vitro is five. 



This method has been modified by various workers so as to use 

 a smaller quantity of blood, to provide anaerobic conditions, etc. 

 For these particulars see Thomson and Thomson (1913), Row 

 (1917), Sinton (1922), and loff (1925). 



