Sorry, this is not my field.

If you need purify the parasite for cultivation, you may try purification using Percoll gradient or isolation in biphasic medium (e.g. NNN/LIT). Alternatively, isolation in conventional medium (Schneider's or RPMI) is possible but with limited sensitivity. 

On the other hand, if you want to detect the parasite, you should try the qPCR or even conventional PCR. I could send you the protocol for both (isolation or PCR).

Best,

Ricardo

The following is an extract from Wyllie and Fairlamb Acta Tropica 97 (2006) 364–369:

"The spleens of infected animals were aseptically removed, weighed and homogenized in Dulbecco’s modified essential medium (D-MEM, Sigma) containing 10% (v/v) foetal calf serum, 20mM l-glutamine and 10mMsodium pyruvate. The homogenate was then centrifuged at 100×g for 5 min at 4 ◦C to remove large cell debris, supernatant collected and centrifuged at 2000×g for a further 10 min at 4 ◦C. The resulting pellet was resuspended in culture medium containing 0.05% (w/v)

saponin and incubated at room temperature for 5 min. Following centrifugation (2000×g, 10 min, 4 ◦C), the amastigote-enriched pellet was washed twice in fresh

culture medium before being resuspended in a final volume of 2 ml. The amastigote suspension was passed through a 23-G needle several times to disperse clumps

prior to counting."

Cells are counted microscopically using a Neubauer counting chamber. A Percoll gradient could be used for additional clean up as suggested above.

Best wishes

Alan Fairlamb