long-term in vitro cultures of Plasmodium falciparum from patient blood can be made. The erythrocyte suspension is washed daily with 50 ml freshly prepared

complete culture medium. The rest of the culture medium was replaced with 200 ml

of freshly prepared complete culture medium in a frequency of once or twice a day

depending on the total number The red blood cells are resuspended twice daily by gentle rotation of the cartridge. Culture method of Li et al (2003) is to be followed.

To understand the rationale underlying media change you should follow BOX 3 in our Radfar et al. protocol published in Nature Protocols (2009) (http://www.nature.com/nprot/journal/v4/n12/abs/nprot.2009.198.html).

To calculate the volume of culture medium needed for each 24 h change we devised the following equation:

V(ml)/24 h = 0.005 (μl RBC pellet)(% parasitemia).

This equation helps calculate the approximate volume of culture medium needed when working with microplates, Petri dishes or culture flasks, to ensure that the infected red blood cells have sufficient medium to survive 24 h. For example, if one wishes to culture a 1,200-μl RBC pellet with a young-stage parasitemia of 20% in a 150-cm2 flask, the volume of medium needed using the above equation will be 120 ml/24 h.

However, for a 1,200-μl pellet containing mature forms, consider the invasion process will occur before 24 h and parasitemia will exceed 20%. Hence, a higher volume of culture medium far from the calculated 120 ml will be required. To deal with this particular situation of departing from mature stages, one may wish to equally distribute the packed culture from each flask into two flasks using fresh erythrocytes and medium to keep a 1% hematocrit in each 120 ml of culture medium, without having to replace the medium twice in 24 h. Optionally, the culture medium should be changed twice in 24 h.

When should we change the media while culturing Plasmodium falciparum?

Well, I would say that depends on how high the parasitemia is, I normally keep them around 5% and change medium everyday (independent of the stage). If the parasitemia is low (around 1% or below) and they are ring stages is ok to skip media change for one day. 

When should we proceed with splitting the culture?

I normally split when the parasites are in late trophs-schizont stages and the parasitemia is above 1.5% (that also depends on the multiplication rate of the strain being used). In my case the multiplication rate is around 6-8, meaning that if one day they are at 1.5% and I don't split them, next day they will be around 9-12% which is the maximum recommended parasitemia for a continuos culture. If the parasitemia is higher than 1.5% I definitely split them otherwise the culture will crash, I adjust the parasitemia depending on what I'm planning to do with them, if needed for experiment you will need rather high parasitemia but if it is just to keep them up and running you can have low parasitemia and therefore you save effort and media (skipping media change when they are rings).

From the protocol you are using it seems you want to get very high parasitemia, you can also have a look to: http://www.mr4.org/Portals/3/Methods_In_Malaria_Research-6th_edition.pdfthis 

Page 7, Growing Plasmodium falciparum cultures at high parasitemia

Essentially you need to lower the hematocrit and change medium twice per day

Thanks a lot to everyone.

Hi Pilar-

I am using Plasmodium falciparum 3D7 for my expt and keeping hematocrit of 1% and planning to maintain parasitemia of 3 %.

I want them for transfection. So any idea how long does it takes to get an appropriate amount of sample.

I have tried culturing twice with 250microL of RBCs and 15% ring stage. But still I am not able to grow the parasites.

For transfection I normally pre-load RBCs with the plasmid and then add parasites to let them reinvade.

I normally prepare a flask with 5ml of medium, 5% hematocrit and 1.5-2% parasitemia (trophozoite stage) I let them reinvade and normally next day I have around 10% parasitemia, then I change medium and wait till they are late stages, then I add all the 250ul of packed cells to around 1ml of plasmid  pre-loaded RBCs and add fresh medium to get 5% hematocrit, then I let them reinvade for 1 or 2 cycles (you have to make sure the parasitemia is not too high) and start drug pressure. 

3D7 normally grows very fast, so if you start with 3% parasitemia in 1% hematocrit you should certainly get at least 10% once they reinvade, if that is not happening maybe something is wrong with them. Do you grow them in candle jar or do you gas them? if using candle jar check there is not any leakage from the box, some parasites are very sensitive to that

I am using candle jar to grow parasites and there is no leakage issue with that.

I took 15 % ring stage parasites in 250microL of RBCs and revived them using nature protocol.My total volume of flask is 40 ml.So accordingly 1 % hematocrite is 400microL.My parasite sample already contained 250 microL . So I added 150microL of freshly washed RBCs and made the total volume upto 40 ml with Complete medium. After 24 hrs I changed the media. However when I tried to check the stages after 40hrs, I am not getting any growth.

When thawed Plasmodium, renewal of culture is two days later. Then the culture medium must be renewed daily. When parasitaemia reached 6%, you must reduce the parasitaemia by bringing new uninfected red blood cells

When starting from a frozen stock it may take more than 1 week to get the parasites growing fine, so maybe you just need to be more patient and give them more time to recover. If after one cycle you don't see any growth maybe there are problems during freezing/thawing procedure.

When I thaw my parasites (200ul, ring, 6-10% parasitemia) I normally don't add any fresh blood, I wait and check them the next day and if they seem to have progress from ring to trophs then I add a little bit of fresh RBCs (20-50 ul) so they have suitable cells to reinvade. Adding 150 ul from the very beginning sounds a lot to me, that would be fine if they are already up and running but in this case you lose quite a lot of parasites during the thawing process, therefore is not such a good idea to split so much immediately. You have to judge when it is time to  split them based on parasitemia increase and life cycle progression, depending on the quality of the freezing/thawing you might need to wait one day or up to a week to get parasites suitable for transfection (they really need to be in top growth conditions)

Did you get the parasites in culture. 

Generally in my experience 3D7 in culture grows 6-8 fold (1% becomes 6-8%). Try to have a synchronized culture after the parasites start growing. 3% trophs (healthy culture)  should give you around 15% parasitemia next day. Change of media every 24 hours will suffice at 1% hematocrit.

If you don't get good multiplicity, try avoiding transfection with that culture

thanks everyone.

I will start again with more precautions and using your inputs.

Generally  culture media should be change every day.But when the parasitemia is mainly schizonts O cells should be added on to the cultures and then split into flask inother to avoid them crashing.

When you start the culture you may use a t25 flask at a start and then bulk them as they grow into a T75 flask and add 25mls of complete medium.

Culture media should be changed everyday.

When you defrost parasites you can leave the culture with same medium during 4 days, there is not problem and the parasites recover faster. After that you shoud dilute and change medium every day.

There are some special circumstances for example:

If you want handle the culture during a weekend you can leave parasitemia in about 0.5 or 0.4 % and 1% of hematocrit and add double amount of medium.

If the parasitemia is high and you need collect merozoites or to study the release of the malaria parasite, to get membranes after release merozoites or in the case to obtain schizonts before releasesing merozoites, you could change medium to the continuous culture three times a day to get the best viability.

The idea is you can handle the parasites as possible according with your time.

Depends on parasitemia. Daily, if maintaining culture at high parasitemia to avoid accumulation of toxic waste that might kill the parasite but also to just provide enough nutrients. If maintaining at low parasitemia eg less than 2%, you can change every other day.

Thank you everyone for your valuable suggestions. :)..

Now my plasmodium culture are growing efficiently.

Hey Guys! I would like to start a malaria imaging project. My lab is an optical based, so little expertise in malaria culturing. I wonder where do you usually purchase blood from? And does the blood has to meet certain infection-free standard, like HIV free?

Thanks!

Andy

In media culture, it is advisable to change the culture media every day but sometimes it depends on how high the parasitemia is especially when it comes to Plasmodium falciparum. For example, if very high parasite/densities (eg >60% parasitemia at high hematocrit like 50%) is needed to be achieved, the medium need to be changed more frequently for better result and to avoid the accumulation of toxic waste that might kill the parasite and if it less than 2% percent then, you can change it every other day.

Reference: Preechapornkul,P., et al (2010). Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3468635/