parasite at dilutions below 0 1 ppe with the levy and ristic conditions 5 0 co 2 in air fluid depth 6 2 mm clones cannot be grown from individual parasites by employing a modified atmosphere 2 o 2 5 co 2 93 n 2 and a lower fluid level 4 0 infected mm propagation of b bovis at very low ppe concentrations down to a single infected cell can be obtained the low ppe concentration propagation is also assisted by frequent removal and replacement of the culture medium above the settled mixture of parasitized and normal rbc 39 s media is replaced once every 24 hours and cells subcultured every 72 hours by making 1 2 dilution with suspension 5 0 pcv of nrbc has been found to be advantagous palmer 1982 parasit 84 567 572 after counting the number of rbc in 1 u03bcl and using the ppe determining the number of infected rbc per u03bcl begin with a multiple of 10 irbc and make serial 1 10 dilutions in culture media so that you isolate 1 irbc in 200 u03bcl of media divide this aliquot in 4 equal parts and dispense into 4 separate wells in the final series.