We grow Plasmodium falciparum 3D7 strain in culture as described by Trager and Jensen in fresh human red blood cells suspended in 2% hematocrit in complete RPMI 1640 medium 500 mL RPMI 1640 supplemented with 25 mM HEPES, 0.2 % glucose, 10µg/ml hypoxanthine, 2 mM L-glutamine, 3µg/ml L-Cystine, 50 µg/mL gentamicin, 0.5% Albumax and incubated at 37°C in a humidified atmosphere of 5% CO2.

RBCs are isolated from whole blood by density gradient centrifugation using reagents called Histopaque and then they are stored in 50% complete growth media at 4 degrees for 2 weeks. We have a problem with non specific staining and false positive in staining non infected red blood cells with Giemsa. When we stain the non-infected RBCs we obtain dark blue spots inside the red cytoplasm of RBC and these spots resemble the shapes of plasmodium falciparum, Therefore we are not able to differentiate between normal red blood cells and red blood cells infected with the parasites.

Our staining protocol is as follows: 1-Allow 1 drop of blood to form on the end of the tube.2- Hold the spreader slide or second microscope slide at a 30-40 degree angle at the end of the slide. 3-Push the spreader slide forward along the length of

the lower slide, while maintaining a constant smooth motion, angle and even

contact. 4- We dry the blood smear using a hiardryer 5- we fix the slide in 100% methanol for 30 seconds. 6- we stain the slide in 10% giemsa solution for 10 minutes.7- we wash the slide with tap water and dry it again with a haridryer

Can you please help us identify the source of non specific staining of our non infected red blood cells