| Summary: | Flow cytometry (FCM) is a powerful method for ploidy determination which has become important because of the increasing use of triploids in aquaculture. Tissue samples for FCM can be biopsied and kept fresh or frozen in a staining solution containing dimethyl sulfoxide (DMSO). Samples can be stored in the stain/DMSO at -80 degreesC indefinitely, or shipped on dry ice to a flow cytometry lab. But ultracold freezers and overnight shipping are not always available, for example, at rural labs and hatcheries. We investigated several methods of preserving FCM samples that do not involve freezing. Three different tissues, gill, mantle, and hemolymph from diploid and triploid pacific oysters, Crassostrea gigas Thunberg, were preserved by different methods, including pre-treatments and different fixatives. Gill was the best tissue for FCM analysis, and ethanol (75%) was the preffered fixative. Hypotonic treatments before fixation promoted nucleus-dissociation needed for FCM. The recommended protocol for preserving gill tissue is to dissect or biopsy a piece Bill tissue (similar to0.5 cm(2)), treat with 0.075 M KCl for 10 min and fix in 75% ethanol that is changed once. Before FCM, the fixed tissue is washed once using a phosphate buffered saline (pH = 6.8) and transferred to a staining buffer containing 10% DMSO. The stained sample is frozen and thawed, votexed. aspiarted, and filtered before analysis. This method can also be used for preserving D-stage larvae and gill tissue samples of other bivalve species.
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