, Volume 77, Issue 4, pp 535-542

Supravital experiments with Pyronin Y, a fluorochrome of mitochondria and nucleic acids

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Summary

The results of fluorochroming living grasshopper spermatocytes, Chinese hamster ovarian tumor cells, and isolated mouse hepatocyte nuclei with Pyronin Y were compared with those obtained in the same material with the mitochondrial probe Rhodamine 123. The patterns of fluorescence obtained with Rhodamine 123 generally coincided with expected distributions of mitochondria except that the fibrous sheaths of the tails of developing and mature spermatozoa were also fluorochromed prominently. After exposure to Pyronin Y, intact cells yielded complex patterns of fluorescence that changed as a function of time: the initial fluorescence resembled that obtained with Rhodamine 123; but after continuous excitation for approximately two minutes, the fluorescence increased by about 60% and became more generally distributed in the cytoplasm and nucleoli of the cells. Since the transition from initial to higher levels of fluorescence could be prevented by pretreatment with RNase, it seems likely that complexes formed between Pyronin Y and RNA were involved in the increase in fluorescence. Transitions from lower to higher levels of fluorescence were not observed in isolated hepatocyte nuclei or in cells with damaged plasma membranes. In these cases, nuclear fluorescence was obtained almost immediately; and the intensity of the fluorescence did not change appreciably with continuous excitation. As in the case of material fluorochromed with Rhodamine 123, the fibrous sheaths of the tails of immature and mature spermatozoa were fluorochromed with Pyronin Y. The results indicate that the demonstration of RNA in supravital preparations may be complicated by the initial reaction of Pyronin Y with mitochondria; and that adequate time should be given for the development of fluorescence attributable to RNA.