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How to Grow a Parasite's Apicoplast Cells in a Culture

Apicoplasts are a type of intracellular organelle present in unicellular parasites that cause severe diseases like malaria, toxoplasmosis and coccidiosis. Organelles are complex structures within a cell that cannot grow and reproduce by themselves. They arose when an ancestor of the parasites engulfed another organism, which was probably related to red algae. Because apicoplasts are not independent cells, they cannot be grown in a culture by themselves. To grow them one needs to grow the parasites. What follows are instructions to culture the red blood cell stage of Plasmodium falciparum, which causes malaria.

Things You'll Need

Parasite stock
RPMI 1640 culture medium
Laminar flow hood
Autoclave
Albumax II serum substitute
D-glucose
Hypoxantine
HEPES
Gentamicin sulfate
Culture flasks
Cell incubator
Red blood cells

Instructions

Culturing Plasmodium falciparum
- 1
  Set the cell incubator to 37 degrees Celsius and adjust the gas composition to 1 percent oxygen, 3 percent carbon dioxide, and 96 percent nitrogen.
- 2
  Autoclave all non-sterile equipment and materials.
- 3
  Working in the laminar flow hood to maintain sterility, prepare the final medium by adding the following to the RPMI 1640 culture medium:
  
  D-glucose to a final concentration of 20 mM.
  
  Hypoxantine to a final concentration of 0.2 mM.
  
  HEPES to a final concentration of 25 mM.
  
  Gentamicin sulfate to a final concentration of 25 mg/L.
  
  Albumax II to a final concentration of 0.5 percent w/v.
- 4
  Distribute the total amount of medium made in the previous step into as many culture flasks as wished and place them on the shaker in the incubator.
- 5
  Once the medium has reached 37 degrees Celsius, add the parasite stock.
- 6
  Change the culture medium every day to keep the parasites well fed and alive.
- 7
  Add red blood cells every other day. They will be infected by the new parasites that are being generated in the culture. If they become too many for a flask, divide its contents into two or more additional ones.

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