1 MATERIALS
1.1 Sterile hood
1.2 Complete medium
1.3 Parasite culture
1.4 Culture gas: 1% oxygen, 3% carbon
dioxide, 96% nitrogen
2 PROCEDURE
2.1 Remove the culture flask carefully from
the incubator without disturbing the sediment cells and move it to the sterile
hood
2.2 Remove the old medium by aspiration
taking care not to remove the cells.
2.3 Smear the stock (10 mL) or large (25 mL)
culture by taking a drop of cells using a sterile pipette. Calculate the
culture parasitemia by Giemsa staining and microscopic inspection (SOP # KLR4)
2.4 Add 37°C complete
medium to the stock culture (10mL) or large culture (25 mL)
2.5 Gas each culture ~30 s using culture gas
mixture. Also gas the complete medium to maintain proper pH. Visually check the
complete medium and cultures daily for traces of bacterial/fungal growth
2.6 Return the culture to incubator unless
dilution is required (see next step). Make sure the culture cap is tightly
closed.
2.7 Each stock and large flask should be
diluted 1:10 when they reach ³ 10% parasitemia. It usually takes 3-4
days after dilution to 1% for the culture to reach ~10% parasitemia again. To
dilute, resuspend the culture and transfer x mL of it into a new flak where:
x=volume of new culture*final
parasitemia required/initial parasitemia
Example 1: x=10*1/10=1 mL
Example 2: x=25*1/10=2.5 mL
2.8 Add fresh blood to the new culture. Grow
cultures @ ~5% haematocrit. Since the blood used is washed and then diluted to
50% using complete medium, to obtain a 5% haematocrit culture, add 1/10th the volume of the new culture fresh washed blood.
Example 1: For a 10 mL stock culture=add 0.5 mL of washed
blood
Example 2: For a 25 mL stock culture=add 2.5 mL of washed
blood
2.9 Add 37°C complete
medium to make of the remainder of the volume of the new culture.
Example 1: For a 1:10 dilution on a 10% parasitemia 10 mL
stock…
1
mL stock culture
1
mL washed blood
8
mL warm complete medium
10
mL total new culture
Example 2: For a 1:10 dilution on a 10% parasitemia 10 mL
stock to a new large 25 mL culture…
2.5
mL stock culture
2.5
mL washed blood
20
mL warm complete medium
25
mL total new culture
2.10 Gas the new cultures for ~30 s using
culture gas and return to the incubator. Make sure the culture flask caps are
tightly closed.