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 eit her new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravi tarly stained with possible long-term storage, using both N and AO. This N/ AO-staining method involves three steps; (1) combination of the target tiss ue or target cells with an equivalent volume of 0.5% solution of new methyl ene 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 dryin g (referred to as fixed-decolorized specimens), and (3) staining with 0.007 % solution of AO for 3 min, followed by washing with Sorensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To ex amine 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 c yclophosphamide. The results indicate that N/AO-stained specimens can be su pravitally observed after long-term storage with the same coloration and co mparable frequencies of micronucleated reticulocytes with a positive respon se 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 n ot to AO-stained specimens, indicative of the preservation of the supravita l feature of N-staining. Taken together these results suggest that the NiAO -staining procedure could offer an additional useful staining tool for the micronucleus test. (C) 2000 Elsevier Science B,V. All rights reserved.