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Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal

Isolates of the marine picocyanobacteria, Prochlorococcus and Synechococcus, are often accompanied by diverse heterotrophic “contaminating” bacteria, which can act as confounding variables in otherwise controlled experiments. Traditional microbiological methods for eliminating contaminants, such as...

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Autores principales: Kearney, Sean M., Coe, Allison, Castro, Kurt G., Chisholm, Sallie W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8888907/
https://www.ncbi.nlm.nih.gov/pubmed/35250937
http://dx.doi.org/10.3389/fmicb.2022.821803
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author Kearney, Sean M.
Coe, Allison
Castro, Kurt G.
Chisholm, Sallie W.
author_facet Kearney, Sean M.
Coe, Allison
Castro, Kurt G.
Chisholm, Sallie W.
author_sort Kearney, Sean M.
collection PubMed
description Isolates of the marine picocyanobacteria, Prochlorococcus and Synechococcus, are often accompanied by diverse heterotrophic “contaminating” bacteria, which can act as confounding variables in otherwise controlled experiments. Traditional microbiological methods for eliminating contaminants, such as direct streak-plating, are often unsuccessful with this particular group of microorganisms. While they will grow in pour plates, colonies often remain contaminated with heterotrophic bacteria that can migrate through the soft agar. Additionally, axenic clones of picocyanobacteria can be recovered via dilution-to-extinction in liquid medium, but the efficiency of recovery is low, often requiring large numbers of 96-well plates. Here, we detail a simple and effective protocol for rendering cultures of Synechococcus and Prochlorococcus strains free of bacterial contaminants while at the same time yielding clonal isolates. We build on the fact that co-culture with specific heterotrophs—“helper heterotrophs”—is often necessary to grow colonies of picocyanobacteria from single cells in agar. Suspecting that direct physical contact between the helper and the picocyanobacterial cells was not necessary for the “helper effect,” we developed a protocol in which the helper cells are embedded in soft agar pour plates, a filter overlaid on the surface, and a picocyanobacterial culture is diluted and then spotted on top of the filter. With this approach, motile contaminants cannot swim to the colonies, and it is possible to obtain the expected number of colonies from a given input (i.e., a Poisson distribution of colonies with an expected value equal to the input number of cells), thus ensuring clonal colonies. Using this protocol, we rendered three strains of Synechococcus, two strains of Prochlorococcus, and 19 new strains of Synechococcus from coastal seawater clonal and free of heterotrophic bacteria. The simplicity of this approach should expand the repertoire of axenic picocyanobacterial strains available for controlled physiological experiments. It will also enable the study of microdiversity in populations of picocyanobacteria by facilitating large-scale isolation of picocyanobacterial clones from a single source, including direct isolation from natural seawater.
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spelling pubmed-88889072022-03-03 Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal Kearney, Sean M. Coe, Allison Castro, Kurt G. Chisholm, Sallie W. Front Microbiol Microbiology Isolates of the marine picocyanobacteria, Prochlorococcus and Synechococcus, are often accompanied by diverse heterotrophic “contaminating” bacteria, which can act as confounding variables in otherwise controlled experiments. Traditional microbiological methods for eliminating contaminants, such as direct streak-plating, are often unsuccessful with this particular group of microorganisms. While they will grow in pour plates, colonies often remain contaminated with heterotrophic bacteria that can migrate through the soft agar. Additionally, axenic clones of picocyanobacteria can be recovered via dilution-to-extinction in liquid medium, but the efficiency of recovery is low, often requiring large numbers of 96-well plates. Here, we detail a simple and effective protocol for rendering cultures of Synechococcus and Prochlorococcus strains free of bacterial contaminants while at the same time yielding clonal isolates. We build on the fact that co-culture with specific heterotrophs—“helper heterotrophs”—is often necessary to grow colonies of picocyanobacteria from single cells in agar. Suspecting that direct physical contact between the helper and the picocyanobacterial cells was not necessary for the “helper effect,” we developed a protocol in which the helper cells are embedded in soft agar pour plates, a filter overlaid on the surface, and a picocyanobacterial culture is diluted and then spotted on top of the filter. With this approach, motile contaminants cannot swim to the colonies, and it is possible to obtain the expected number of colonies from a given input (i.e., a Poisson distribution of colonies with an expected value equal to the input number of cells), thus ensuring clonal colonies. Using this protocol, we rendered three strains of Synechococcus, two strains of Prochlorococcus, and 19 new strains of Synechococcus from coastal seawater clonal and free of heterotrophic bacteria. The simplicity of this approach should expand the repertoire of axenic picocyanobacterial strains available for controlled physiological experiments. It will also enable the study of microdiversity in populations of picocyanobacteria by facilitating large-scale isolation of picocyanobacterial clones from a single source, including direct isolation from natural seawater. Frontiers Media S.A. 2022-02-16 /pmc/articles/PMC8888907/ /pubmed/35250937 http://dx.doi.org/10.3389/fmicb.2022.821803 Text en Copyright © 2022 Kearney, Coe, Castro and Chisholm. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Kearney, Sean M.
Coe, Allison
Castro, Kurt G.
Chisholm, Sallie W.
Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal
title Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal
title_full Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal
title_fullStr Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal
title_full_unstemmed Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal
title_short Filter Plating Method for Rendering Picocyanobacteria Cultures Free of Heterotrophic Bacterial Contaminants and Clonal
title_sort filter plating method for rendering picocyanobacteria cultures free of heterotrophic bacterial contaminants and clonal
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8888907/
https://www.ncbi.nlm.nih.gov/pubmed/35250937
http://dx.doi.org/10.3389/fmicb.2022.821803
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