Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates.

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0.65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28 degrees C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0.6% agarose overlay containing G418 as the selective drug, on top of a 0.8% agar base. Transformation efficiencies were as high as 10(-5) cells per microgram of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of 'shotgun transformation' to generate libraries of T. cruzi recombinants.