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: Packard, Benjamin H
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 and other related dinoflagellate algae have been implicated as causative agents of fish kills. One objective of this study is to establish culture isolates of Pfiesteria and Pfiesteria-like organisms for future research experiments to be conducted by members of the scientific community. Water and mud samples will be collected from regions where Pfiesteria and Pfiesteria-like organisms occur, single cells will be isolated, and the established cultures will be maintained. These culture isolates will be deposited in the Provasoli-Guillard National Center for Culture of Marine Phytoplankton and will be available to the scientific community for research experiments. The second objective of this study is to develop a method for cryopreserving strains of Pfiesteria and Pfiesteria-like organisms. Cryopreservation is the process of freezing cells without damage, maintaining the cells in a frozen state at liquid nitrogen temperatures (-196 uC), and producing actively growing cells once the frozen cells have been thawed. There are several advantages for maintaining frozen cells. There is no routine handling of cells and therefore the possible exposure to toxins is avoided. Cryopreservation reduces labor and material costs for long term maintenance. Also, cell lines sometimes mutate during ordinary maintenance, but frozen cells at liquid nitrogen temperatures are thought to be free from any possible mutations because all biological activity is suspended due to the extremely cold temperature. Methods have been developed for cryopreservation of many micro-organisms, including algae, and the objective is to modify these methods so that Pfiesteria and Pfiesteria-like organisms can be successfully cryopreserved.


Scientists and governmental employees in the areas where Pfiesteria and Pfiesteria-like organisms occur will collect samples of mud and water. The samples will be sent by overnight courier and Pfiesteria and Pfiesteria-like organisms will be isolated using a fine micropipette. In addition, the samples will be enriched by adding Rhodomonas, a food item of Pfiesteria and Pfiesteria-like organisms. For cryopreservation, a computer-modulated controlled-rate freezing unit will be used to establish several steps, each with a different cooling rate. Different cryoprotectants (i.e., substances that protect cells from damage during freezing) will be tried. Upon completion of the freezing procedure, the cells (in cryo-vials) will be placed in a liquid nitrogen storage tank. Frozen cells will be thawed and viability will be assessed. Variations in (a) the steps and rates of freezing, (b) cryoprotectants, and (c) thawing will be tried to achieve success and to maximize % viability.

Expected Results:

A substantial number (e.g., greater than 50) of single-cell isolates should be established and made available to the scientific community; these strains should allow scientists to determine the species diversity as well as genetic diversity of Pfiesteria and Pfiesteria-like organisms. A successful method for cryopreserving Pfiesteria and Pfiesteria-like organisms should be developed.

Publications and Presentations:

Publications have been submitted on this project: View all 2 publications for this project

Journal Articles:

Journal Articles have been submitted on this project: View all 1 journal articles for this project

Supplemental Keywords:

CCMP, culture collection, marine, phytoplankton, phycology, protist, protozoology., 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

Progress and Final Reports:

  • 1999
  • 2000 Progress Report
  • 2001 Progress Report
  • Final Report