A RAPID AND PRECISE TECHNIQUE FOR ENUMERATION OF RESTING CYSTS OF ALEXANDRIUM SPP (DINOPHYCEAE) IN NATURAL SEDIMENTS

A new method is described that uses the fluorochrome primuline and epi fluorescence microscopy for precise enumeration of dinoflagellate cyst s in natural sediments. Alexandrium tamarense (Lebour) Balech resting cysts obtained in laboratory culture were fixed with glutaraldehyde an d treated with methanol. The cysts were stained using nine fluorochrom es under identical procedures to find those suitable for enumerating c ysts. Four fluorochromes, acrilflavine, calcofluor white M2R, nile red and primuline, were found to provide satisfactory results in terms of high stainability and fluorescence intensity. Methanol treatment afte r fixation was necessary for high stainability. The four fluorochromes were then examined for their applicability to enumerate naturally occ urring cysts in Hiroshima Bay sediments. Primuline proved to be superi or to all other dyes examined, providing higher counts of cysts. Primu line-stained cysts exhibited an intense yellow-green fluorescence unde r blue-light excitation which highlighted the cysts from background pa rticles. It was confirmed that all cysts found by conventional light m icroscopy were clearly stained with this dye. Moreover the number of c ysts obtained by the primuline-staining method was two or more times h igher than that obtained by normal light microscopy, which required fi ve times as long for observation. The primuline-staining method reveal ed that density gradient centrifugation using colloidal silica (Ludox TM) for separation and concentration of natural cysts underestimates t he number of cysts in sediments unless the detrital material around th e cysts is removed. Using the primuline-staining method, it is possibl e to observe rapidly large amounts of sediment and thereby obtain more reliable estimates of cyst abundance. Primuline also stained cysts of other flagellates; i.e. Protoperidinium spp., Scrippsiella spp., Pyro phacus sp., and Chattonella. The primuline-staining technique may repl ace conventional methods for enumerating dinoflagellate resting cysts.