Trypanosoma cruzi Y reference strain is found in many laboratories under at least two highly distinct genotypes, A and B corresponding to the 'discrete typing units' T. cruzi IIb and T. cruzi IId, respectively. Previous work has reported reversible switches between these genotypes according to the culture media used in the experiments: genotype A would be associated with blood-enriched culture media, while genotype B would be associated with blood-free culture media. We tried to reproduce this observation, but used a different cloning method of individual organisms. Our cloning was verified visually under the microscope, while the previous studies relied on a cloning by dilution only. The subclones so obtained were submitted to long-term exposure to both media, and no change was observed in isoenzyme and random amplified polymorphic DNA genotypes. The discrepancy is probably explained by the cloning method: clones obtained from the previous method (dilution and plating) could come from several parasite cells while only one cell generates a clone when micro-manipulation is used.