Cytofluorometry as a method for the differentiation of trypanosomes.

The DNA binding guanine specific antibiotic, chromomycin A3, has been evaluated for fluorescence intensity measurements of T. cruzi, T. brucei brucei and T. musculi. Optimal fixation and staining conditions have been determined. The fluorometry was performed with a microscope photometer equipped with electronic systems for short time excitation of 7 milliseconds and operation control. The trypomastigote bloodstream forms of these species have a different chromomycin specific DNA content. The total DNA content of T. cruzi was 2.1-fold higher than for T.b. brucei and 2.3-fold higher than for T. musculi. The nuclear DNA content also was higher in T. cruzi. The nuclear DNA values were recorded to be 1.6-fold greater than in T.b. brucei and 2.0-fold greater than in T. musculi. The amount of the kinetoplast DNA of T. cruzi was shown to be 3.2-fold higher than in T. musculi and 11.7-fold higher than in T.b. brucei. The higher total DNA of T.b. brucei in relation to T. musculi was based on the nuclear values because the content of the kinetoplast DNA of T.b. brucei was 3.7-fold smaller than of T. musculi. The kDNA comprised 25% in T. cruzi, 18% in T. musculi and only 4% in T.b. brucei of the total amount of the chromomycin specific DNA. The chromomycin fluorescence intensities of the DNA of trypanosomes were subjected to a statistical model of discriminant analysis. It was possible to get perfect separation of the three trypanosome species. The hit rate was 100%.