MEPS 237:65-78 (2002)  -  doi:10.3354/meps237065

ABSTRACT: A method to isolate cells of the toxic dinoflagellate Alexandrium fundyense from seawater samples using magnetic beads was evaluated to determine whether it could be used to obtain species-specific physiological measurements. Two isolation procedures were tested. The direct technique used an Alexandrium-specific primary antibody coupled directly to magnetic beads, which were then bound to the target cells. With the indirect technique, the primary antibody (Ab) was bound to the target cells, and this Ab-cell complex was then exposed to the beads. Four different bead types, varying in size and coating, were evaluated. The bead isolation method was tested on live cells and on cells preserved with either 0.23 N sulfuric acid or 4% formalin. Under optimal conditions using either live or preserved cells, ca. 80% recovery was achieved when the indirect technique was applied with either M-280 Streptavidin or M-280 sheep anti-mouse beads. However, with the direct technique, the highest recoveries were only ca. 20% with live samples and ca. 50% with acid-preserved cells. After bead isolation, ca. 70% of the recovered live cells were intact; 30% were completely or partially broken. A variety of post-isolation measurements were conducted on the separated cells, including nucleic acids, total proteins, chlorophyll a (chl a), C-2 toxin, saxitoxin (STX), neosaxitoxin (NEO), organic carbon (C) and nitrogen (N), and ¹³C/¹²C and ¹⁵N/¹⁴N. These measurements were normalized to the number of intact cells. For live samples, there were no statistically significant differences between the control cells and the bead-recovered cells for cell quotas of nucleic acids, total proteins, chl a, STX, C-2 toxin, and NEO. Similarly, no differences were found in these parameters between the live samples and the acid-preserved samples, except for the nucleic acid measurements where the acid interfered with the measurements. However, there were significant differences between the live cells and formalin-preserved, bead-recovered cells for all parameters measured. This was not due to the bead isolation procedure but to the preservative. Significant C, N, C/N, ¹³C/¹²C, and ¹⁵N/¹⁴N differences between live cells and bead-recovered cells were introduced by the immunomagnetic separation protocols, due to the chemical and isotopic content of the beads. These artifacts need to be removed if monospecific elemental and isotopic abundance measurements are to be attempted using this isolation method. Overall, immunomagnetic cell sorting is potentially a valuable tool for taxon-specific isolation, allowing physiological, chemical, and isotopic characterization of naturally occurring phytoplankton populations. However, each measurement must be evaluated to insure that the cell components do not change due to the isolation procedure.

KEY WORDS: Alexandrium fundyense · Dinoflagellate · Immunomagnetic isolation · Magnetic beads · Saxitoxin · ¹⁴C uptake · Chlorophyll a · DNA · RNA · ¹³C/¹²C · ¹⁵N/¹⁴N · Carbon · Nitrogen · Protein