Sample dilution series for parasite cloning (assuming 5 % hematocrit and 1%
parasitemia)
1.
2.
3.
4.
5.
6.
7.
5 % hematocrit= 5 x 108 cells per ml
at 1% parsitemia = 5 x 106 parasites per ml of your culture
100 μl of culture diluted in 10 ml media/blood = 5 x 104 parasites per ml
100 l of #3 diluted in 10 ml of media/blood = 5 x 102 parasites per ml
100 l of #4 diluted in 10 ml of media/blood = 50 parasites per ml
1 ml of #5 diluted in 19 ml of media/blood = 50 parasites total
The 20 mls from #6 can be aliquoted into a 96 well plate at 200 μl per well.
The 96 well plate should be put in a gas chamber, and the chamber gassed and sealed.
Change the media one a week for the first two weeks, every second day after that. When
changing the media, use replacement media in which you have added fresh red blood
cells. For example, add 100 μl of fresh RBCs to 10 mls of replacement media. Parasites
should be visible around 3 weeks after plating.