Final Report: Culturing and Cryopreserving Pfiesteria-like Organisms

EPA Grant Number: R826793
Title: Culturing and Cryopreserving Pfiesteria-like Organisms
Investigators: Andersen, Robert A.
Institution: Bigelow Laboratory for Ocean Sciences
EPA Project Officer: Hiscock, Michael
Project Period: October 1, 1998 through September 30, 2001 (Extended to September 30, 2002)
Project Amount: $275,002
RFA: Ecology and Oceanography of Harmful Algal Blooms (1998) RFA Text |  Recipients Lists
Research Category: Water Quality , Harmful Algal Blooms , Water , Ecosystems


Pfiesteria piscicida, Pfiesteria shumwayae, and related organisms (Pfiesteria-like organisms) have been implicated as causative agents for fish kills and human health problems. One objective of this study was to establish cultures of these organisms so that other research scientists could conduct experimental studies to investigate these implications. Samples were collected from areas where Pfiesteria-like organisms occurred or were suspected to occur, and single cells were isolated using the micropipette method. The isolates were deposited in the Provasoli-Guillard National Center for Culture of Marine Phytoplankton. The second objective of the study was to develop methods for cryopreserving the cultures. It is known that microorganisms sometimes change in culture after prolonged periods of serial transferring from old cultures to new medium. Furthermore, serial transfer requires handling of these potentially dangerous algae every few weeks, and human error can occur (e.g., mislabeled tubes, contamination, breakage, or spillage). Finally, workers could be exposed to toxins when making serial transfers, and the level of toxins produced by cells may change during prolonged periods of serial transfer. Cryopreservation involves freezing the cells in such a manner that, when they are thawed, they are alive and growing. Cryopreserved cells are maintained at a temperature of at least -155°C, or colder, by placing the frozen cells in storage tanks cooled with liquid nitrogen. Once frozen, the cells undergo no change, there is no handling, toxicity remains unchanged, and toxin exposure is eliminated.

Summary/Accomplishments (Outputs/Outcomes):

During the project, I isolated 57 strains of Pfiesteria and Pfiesteria-like organisms, and I deposited these in the Provasoli-Guillard National Center for Culture of Marine Phytoplankton (CCMP). Collection sites for these strains included Chesapeake Bay, the Neuse River (North Carolina), and the Wilmington River (Georgia). In addition, nine strains were isolated from ship ballast water of the Alicante Carrier, which left the Port of Moin, Costa Rica, and docked in the port of Philadelphia, Pennsylvania. The ship’s log states that the ballast water was obtained from the Port of Moin. If so, these are the first reports of Pfiesteria-like organisms from Costa Rica. However, if for some reason the ship also obtained ballast water while traversing the Chesapeake Bay, the organisms may have originated from the Chesapeake Bay. The CCMP, of which I am Director, also accepted one Pfiesteria piscicida strain from Pat Tester’s research group (National Oceanic and Atmospheric Administration), one Pfiesteria piscicida strain from Karen Steidinger’s research group (state of Florida), and one Pfiesteria shumwayae from the Virginia Institute of Marine Sciences research group during the project. Finally, I isolated approximately 20 additional strains that have not yet been deposited in the CCMP, including one strain from Japan.

We have successfully cryopreserved 31 strains, and we are in the process of freezing the remaining strains. The first, general method we developed is not complicated, but it is labor intensive, and it takes considerable time to grow, filter, and freeze the cells. We are currently trying a modified protocol that induces cyst formation, and this appears to be an even more successful method. The new method was tried after discussions with colleagues at the Virginia Institute for Marine Sciences, who developed the cyst formation method for other purposes.

We carried out an extensive examination of the salinity tolerance for 62 strains. Knowledge of salinity tolerance is important in selection of future sampling sites, and it is of value in the cryopreservation process. The strains tolerated salt concentrations from near freshwater to full seawater, and many strains survived at salinities of 50--60 psu. Strains isolated from the same area had similar salinity tolerances, with those from the Chesapeake Bay and Neuse River systems tolerating lower salinity water, while those from Georgia tolerated higher salinity water. A resulting publication is listed below.

We began preparing a manuscript to be submitted to the Journal of Phycology that described our general cryopreservation freezing method for Pfiesteria. However, we are now trying to optimize the new method that induces cyst formation, and manuscript preparation has stalled until we have finalized this procedure. It should be noted that we have already circulated the cryopreservation method to other Pfiesteria researchers so that they could begin using the method if desired. Our Web site, Exit , gives details of the general method that was used for strains already cryopreserved.

We have also examined the temperature tolerance of 62 strains using a gradient block and 10 replicates for each strain. Results show that the strains can, in general, tolerate temperatures between 4-5°C and 30-35°C, with one strain surviving at 38°C. Although these strains come from warmer regions of the United States, and therefore they may be adapted for temperatures no colder than 4°C, the results may also offer an explanation for why Pfiesteria does not occur in regions, such as Maine, where freezing temperatures occur.

In summary, the primary goals of the grant were to isolate new strains of Pfiesteria and Pfiesteria-like organisms and to develop methods for cryopreserving this organism. We have achieved both goals. In addition, the results of our salinity and temperature tolerance studies provide autecological data for Pfiesteria and Pfiesteria-like organisms. These data may aid managers in their efforts to understand the possible spread of Pfiesteria to other regions, whether by ballast water or other means.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 2 publications 1 publications in selected types All 1 journal articles
Type Citation Project Document Sources
Journal Article Sullivan BE, Andersen RA. Salinity tolerances of 62 strains of Pfiesteria and Pfiesteria-like heterotrophic flagellates (Dinophyceae). Phycological Research 2001;49(3):207-214. R826793 (2001)
R826793 (Final)
  • Abstract: Wiley
  • Supplemental Keywords:

    algae, Atlantic Coast, Chesapeake Bay, Costa Rica, Center for Culture of Marine Phytoplankton, CCMP, cryopreservation, culture strains, Pfiesteria, salinity and temperature tolerance,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Oceanography, algal blooms, Ecological Risk Assessment, cryopreserving, fish kills, culture collection, phytoplankton, National Center for Marine Phytoplankton, pfiesteria, dinoflagellate, laboratory studies

    Relevant Websites: Exit

    Progress and Final Reports:

    Original Abstract
  • 1999
  • 2000 Progress Report
  • 2001 Progress Report