2000 Progress Report: Culturing and Cryopreserving Pfiesteria-like OrganismsEPA 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 Period Covered by this Report: October 1, 1999 through September 30, 2000
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
Objective:Pfiesteria piscicida and related organisms (Pfiesteria-like organisms) have been suggested to be involved in fish kills and human health problems. One objective of this study is to establish cultures of these organisms so that other research scientists are able to conduct experimental studies. Samples are collected from areas where Pfiesteria-like organisms occur or are suspected to occur, and single cells are isolated using the micropipette method. The isolates are deposited in the Provasoli-Guillard National Center for Culture of Marine Phytoplankton. The second objective of the study is to develop methods for cryopreserving the cultures. Microorganisms sometimes change in culture after prolonged periods of perpetually transfer from old culture to new medium. Furthermore, perpetual transfer requires handling every few weeks, and human error can occur during this handling (e.g., mislabeled tubes, contamination, breakage or spillage). Finally, workers might be exposed to toxins when making perpetual transfers, and the level of toxins produced by cells may change during prolonged periods of perpetual transfer. Cryopreservation involves freezing the cells in such a manner that when they are thawed, they will still be 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.
Progress Summary:We have isolated five strains of Pfiesteria piscicida, and we have established two axenic (bacteria-free) strains of Pfiesteria piscicida from those five isolates. We have isolated 66 strains of Pfiesteria-like organisms, and 43 of these are officially deposited in the Provasoli-Guillard National Culture Center of Marine Phytoplankton (CCMP). The remaining 23 are available to scientists by contacting the CCMP, but they are not officially deposited in the collection. The reason for not officially depositing the additional 23 strains is they have not been cryopreserved yet. If cryopreservation is successful, they will be officially added to the CCMP. It may be significant to note that nine of the isolates were recently (summer 2000) isolated from the ballast water of a ship. The ship originated in Puerto Rico, obtained its ballast water at port or from the Caribbean Sea, and sailed to Philadelphia. This result demonstrates a more widespread occurrence of Pfiesteria-like organisms, and it demonstrates the ability of ships to distribute this organism.
We have successfully cryopreserved 31 of the isolates, including four of the five Pfiesteria piscicida strains. Many of the remaining strains are likely to cryopreserve successfully as well. The method we developed is not complicated, but it is labor intensive, and it takes considerable time to grow, filter and freeze the cells.
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. Furthermore, other scientists may find the salinity tolerance of value. In summary, the strains tolerated salt concentrations from near freshwater to full seawater, and many strains survived at salinities of 50 to 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.