A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.