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Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds
The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource re...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MyJove Corporation
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352255/ https://www.ncbi.nlm.nih.gov/pubmed/28117830 http://dx.doi.org/10.3791/55064 |
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author | Hockett, Kevin L. Baltrus, David A. |
author_facet | Hockett, Kevin L. Baltrus, David A. |
author_sort | Hockett, Kevin L. |
collection | PubMed |
description | The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin. |
format | Online Article Text |
id | pubmed-5352255 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MyJove Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-53522552017-04-04 Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds Hockett, Kevin L. Baltrus, David A. J Vis Exp Infection The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin. MyJove Corporation 2017-01-14 /pmc/articles/PMC5352255/ /pubmed/28117830 http://dx.doi.org/10.3791/55064 Text en Copyright © 2017, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ |
spellingShingle | Infection Hockett, Kevin L. Baltrus, David A. Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds |
title | Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds |
title_full | Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds |
title_fullStr | Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds |
title_full_unstemmed | Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds |
title_short | Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds |
title_sort | use of the soft-agar overlay technique to screen for bacterially produced inhibitory compounds |
topic | Infection |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352255/ https://www.ncbi.nlm.nih.gov/pubmed/28117830 http://dx.doi.org/10.3791/55064 |
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