For the culture media, normally I use RPMI 1640 supplemented with hypoxanthine , glucose, HEPES, albumax II, NaHCO3 and gentamycin (antibiotic) 

For culture condition, i do the same with you.

Dear Jutharat,

My medium already has HEPES in it. Rest all mentioned by you I add except glucose. Can that be the possible reason for the growth failure? Does your culture work fine with the mentioned conditions??

 I think glucose is important for parasite growth. After i use the condition that i mentioned you, i can continuous culture Plasmodium falciparum.

You do not provide any volumes so I cannot say much about it. But you can find all these type of protocols with MR4: 

https://www.beiresources.org/portals/2/MR4/Methods_In_Malaria_Research-6th_edition.pdf 

I would at least increase your hematocrit to 2.5 or 5% and change your medium daily for now. Only when your parasite density is increasing you dilute with fresh (washed) RBCs (or you grow them untill the desired parasite density, I don't know what the purpose is of your culture). I have never added glucose to my culture medium and they grow like crazy, so I don't think that is the problem. I did use RPMI 1640 Hepes L-gluthamine, albumax II plus human heat-inactivated serum, hypoxanthine and I used gentamicin (maybe your antibiotic antimycotic 'mixture' is causing problems?). 

Good luck

Hi,

As Esmee, I think the problem might come from your antibiotic mixture, as some antibiotics inhibit the growth of Plasmodium. What antibiotics and fongicide contain your mixture ? 

I add 1% antibiotic-antimycotic mixture (10,000 IU/mL penicillin, 10,000 ug/mL streptomycin and 25 ug/mL amphotericin B) to the culture media. Should I decrease its concentration in the media? Or use other antibiotic, say gentamycin as mentioned by Esmee?? Thank you..

Yes I recommend you to change the antibiotic and use gentamicin that is usually used for Plasmodium. If you want to be sure that the problem comes from the antibiotic take a part of your culture and try to cultivate it without antibiotics (if you're carefull you shouldn't have any contamination. If the growth of your parasites increase then you will have a clear answer ;). Good luck !

Hi dear,

Try with high volume of hypoxanthine (almost double) for some time about one month, if successful than return to normal conc. of hypoxanthine. Also used culture disc instead of culture flask. 

Thanks.

Hi Naisargee

As they told you, I think the antibiotic can affect the growth of the parasites. We use only gentamicin (0.016g / L). I think that if you do not have many problems of contamination, you can not use any type of antibiotic.

Another thing I can see is that it can be changed in the hematocrit (we use 5%), the atmosphere of gases we use has 02 and C02 to 5% and erythrocytes of type A + increase the invation.

I hope it helps you.

Good luck !

yes the crucial step for the growth is the antibiotic concentration I think the gentamicin only is quite no need for anti fungal please if possible do different concentration of gentamicin until the growth occurs every parasite have different behavior invitrO., Also you increase the initial parasitaemia as 5%, as Adriana say blood group of  A+ is ok.

I hope this point to help you

https://malariajournal.biomedcentral.com/articles/10.1186/s12936-015-0663-x

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC15536/

Your problem may be the gas, which is 5%CO2. Most of the labs normally use a gas mix of 5%CO2, 5%O2 and 90%N2. There are labs that use 5% CO2, but one would need a parasite strain adapted to that condition.

Other issue could be albumax II lot. Some lots of albumax do not support Plasmodium growth. You can test that by using anoth lot of albumax or 10% human serum.

Better start culturing without antibiotic or just use the 10mg Gentamycin in 1L media. Rest is all ok. I am culturing with this way from 10 months and my culture is running well. Other antibiotics won't work well with plasmodium culture.

Hello everyone

Sorry to add a question instead of giving an answer

Thank you for your brilliant answers. indeed, I have almost the same problem as Patel with my cultures. Since 2 months, parasites don't grow, yet they were growing well. But I use only Gentamycin at 0.02g/L as antibiotic and 3% hematocrit.

Please can you advice me something?

Looking forward to hear from you

Hello everyone..

Rodrigue, according to the struggles that I have faced for culturing the parasites, I would suggest you to keep the RBC donor constant and not to use RBCs older than 1 week. RBCs are a crucial factor for P. falciparum culture. Certain RBCs, if not compatible, will lead to crashing of culture if added. This factor has caused a drastic change in my culture rates. Hope this helps you !!! Good luck :)

Hello everyone,

I am having the same problem as Rodrigue. Since almost three months I can not make the cultures grow (3D7 strain, 1.5%HTC). The media I use the same as you'll, with hypoxanthine, glucose, inactivated human serum, and I use RBCs A+ (actually mine). But I do not understand if it is because the cultures were old (i started last year, november), or the gas concetration (I use the candle jar method), or one of the reagents in the media.

Thanks!

Hello maria...

it is much likely that your rbc are not compatible with the culture. also, was your culture stock stored in liquid nitrogen? p. falciparum do not survive in -80. hope this myt help u. good luck !

Hello Naisargee,

Before they were growing fine with my blood )i have been working with them since 18 months) and the problem started three months ago. I stored stock stabilates at -150 (as my PI did before)

Hello everyone,

Dear Maria, as you know, I had the same problem and I found it was the candle jar the problem because it let out the gas. So I think it could be also the cause of your situation. For this, I advice you to culture your parasites directly in incubator (without candle jar) by making sur that there is CO2. Please try and let us see how it will evolve then we can process another resolution

thank

Dear Rodrigue,

Thank for your suggestion, I will try it to do so and come back to you

You are welcome Maria. So what about your culture? your parasites grow well now?