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Use of micromanipulation and "feeder layers" to clone the oyster pathogen Perkinsus marinus
D Bushek1, R A Holley, K S Reece
1Baruch Marine Field Laboratory, Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Georgetown 29442 USA. bushek@sc.edu
Abstract:
Genetic and biochemical characterization of microbes often requires the use of clonal cultures. A method to clone the oyster parasite Perkinsus marinus is described. Individual cells are isolated via micromanipulation and maintained above an actively proliferating "feeder layer" of P. marinus on a 0.45-microm membrane. Extracellular products released from the proliferating feeder layer can diffuse across the membrane and bathe the isolated cell, stimulating it to proliferate. The method is relatively simple and should be applicable to most protists that can be cultured in the laboratory.
Insights
A new method simplifies cloning of microbes like the oyster parasite Perkinsus marinus. This technique uses a feeder layer to stimulate isolated cells, enabling clonal culture for research.
Area of Science:
- Marine biology
- Microbiology
- Parasitology
Background:
- Clonal cultures are essential for microbial genetic and biochemical studies.
- Culturing protists, especially parasites like Perkinsus marinus, can be challenging.
Purpose of the Study:
- To describe a novel and accessible method for obtaining clonal cultures of the oyster parasite Perkinsus marinus.
- To provide a technique applicable to a wide range of culturable protists.
Main Methods:
- Individual Perkinsus marinus cells were isolated using micromanipulation.
- Isolated cells were cultured on a feeder layer of P. marinus.
- Cells were supported by extracellular products diffusing through a 0.45-microm membrane.
Main Results:
- The described method successfully stimulated proliferation of isolated P. marinus cells.
- The technique provides a means to establish clonal cultures of this oyster parasite.
- The method is noted for its relative simplicity.
Conclusions:
- This micromanipulation and feeder layer technique offers a practical approach to clonal cell culture.
- The method has potential broad applicability for culturing various laboratory-grown protists.
- Facilitates genetic and biochemical characterization of hard-to-culture microbes.