A comparison of various methods for the determination of viability of parasitic flagellates

To find out a simple, rapid, accurate, and reliable viability assay for in vitro anti-protozoa drug testing, a comparison among the various methods for the determination of flagellate viability was made. Micro-colony forming efficiency up to 90% for three different flagellates was obtained by the agar plate method. Macro-colony forming efficiency obtained by the culture tube method was generally lower (20-45%). Viability determined by trypan blue exclusion correlated closely with viability determined by micro-colony count (r = 0.94). The growth of the remaining parasites survived from serum treatment was not inhibited while that from metronidazole pretreatment was inhibited. The standard regrowth assay based on the 48-hr cultivation could be applied only for those survival not inhibited. The inhibitory effect of metronidazole on parasites was further testified by the longer generation time for metronidazole-pretreated flagellates in comparison with that for control (p less than 0.001). If the inhibitory effect of a drug is involved in the mechanisms against parasites, only the trypan blue exclusion and the micro-colony count can give an accurate and reliable viability determination. Moreover, the simple and rapid diagnosis provided by trypan blue exclusion is especially useful for the in vitro anti-protozoa drug susceptibility test.