I interested

I think it depends a lot on the species and strain of Leishmania that you are culturing. Are you sowing under sterile conditions directly in Novy-MacNeal-Nicolle (NNN) and Schneider culture media with 10% fetal bovine serum?

Dear Souza thank you for your response, yes i used complete media of RPMI with (10% FBS, 1% antibiotics and 1% HEPES).

If the rate of growth is slow in fresh medium, you can use some old used culture supernatant with fresh medium. Worked for me.

Dear Obaid,

You are culturing L. tropica no? Give me more info. What is the antibiotic you use and incubation temperature? Are those from patient isolates or from cryopreserved samples? Have you observed those white colonies under inverted mic? Any fungal contaminations?

Dear Bhagya,

I'm culturing L.tropica ,isolated from patients, using 1% Streptomyacin, a white cluster or colony formed when i analysed after 24 to 48h with naked eye but there is still an active leishmania when i analysed on inverted microscope but with limited numbers of parasite. I also obtain free contaminated culture from the patient isolates but not going to riched however the using protocol is same for all the culturing and sub culturing is same.

You face this problem with all patient isolates or only for some isolates? Haven't you done sub-culturing and mass culturing with the protocol you mentioned for patient isolates before?

The contamination problem is accur in some isolates, while culture poor mean not going rich is accur in every one.

The slow growth of the parasite may be due to the concentration of antibiotics. You must try with different concentrations of antibiotics to standardized it in your lab conditions.

Moreover, you can try maintaining sterile conditions for culture to minimize the chances of contamination as you observed white cluster in your culture.

And for this you can try using gentamycin instead of streptomycin or with it, in your culture medium as per requirement.

I hope you got your answer..........

Thanks and Good luck.....

Agree with Deepak's answer. You can reduce concentration of antibiotics from 1% to 0.1%. But be careful with contaminations when using lower concentrations.

You can use gentamycin or penstrep.

Obaid Hayat thank you for your response. First of all, check any kind of contamination in the culture. If okay, spin down the parasites at 1500 rpm for 10 minutes at room temp. Collect the supernatant (used media). Wash the parasites with fresh incomplete medium twice. Mix the pellet with 1 ml fresh complete medium, and add100 ul culture to 4.5 ml fresh complete medium. Now add 500 ul of collected old medium to this fresh culture. and keep your flasks horizontally in the incubator. I hope will be helpful.