Flow cytometry application in marine phytoplankton study: a case study investigating effects of formalin preservation on phytoplankton count and cell integrity

Flow cytometry has been used in oyster feeding studies at Delaware State University for counting and sorting of marine micro-algal cells to determine the filtration activities of American oyster (Crasostrea virginica). This study was conducted to develop a simple protocol for accurate quantification of several cultured algae, using a known quantity of fluorescent microspheres as an internal calibration standard. While using flow cytometry to analyze algal samples in filtration studies on the oysters (C. virginica), we observed shifts in algae populations in both preserved and unpreserved samples over time. Because of these shifts, we felt it was important to determine the most efficient way to quantify algae accurately used in the filtration study. Two algal species, Isochrysis spp. (TISO) and Tetraselmis chui (PLY-429) cultured in F/2 media under the laboratory conditions at 12/12 hour light/dark cycles at 24°C room temperature at a set light intensity (130 umol), were used in this study. In order to determine the effects of preservation, via formalin we examined formalin-preserved and unpreserved phytoplankton samples both with and without known concentrations of fluorescent counting beads. Cell counts of cultured algal species for each treatment were determined using flow cytometry. The technique for counting and calibrating equipment were discussed. Significant (P < 0.05) differences were noted in between formalin-preserved and unpreserved samples as well as over time in 50% of the experimental trials out of four trials conducted for each algae species. Unpreserved samples showed reduced cell counts after 6 days in 75% of trials. Formalin-preserved samples showed significant reductions (P < 0.05) in cell counts after 3 days. Samples preserved with formalin and samples having formalin and beads showed lower algae counts immediately after the first day samples analyzed. We observed a significant (P < 0.05) reduction of fluorescent intensity over time. This reduction was most visible in the formalin-preserved samples with and without beads on the sixth day of sampling. Fluorescent counting beads were also affected by seawater. This study is intended to provide information on the applications of flow cytometry in phytoplankton research, and basic protocols needed to perform measurements on formalin-preserved and unpreserved samples. The methods may need to be modified for other applications.