Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains
ABSTRACT We previously showed that autoclaving in preparing agar media is one of the sources of hydrogen peroxide (H2O2) in the medium. This medium-embedded H2O2 was shown to lower the total colony count of environmental microorganisms. However, the critical concentrations of H2O2 detrimental to col...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-12-01
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| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.03336-22 |
| _version_ | 1798003808027017216 |
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| author | Motoyuki Watanabe Kensuke Igarashi Souichiro Kato Yoichi Kamagata Wataru Kitagawa |
| author_facet | Motoyuki Watanabe Kensuke Igarashi Souichiro Kato Yoichi Kamagata Wataru Kitagawa |
| author_sort | Motoyuki Watanabe |
| collection | DOAJ |
| description | ABSTRACT We previously showed that autoclaving in preparing agar media is one of the sources of hydrogen peroxide (H2O2) in the medium. This medium-embedded H2O2 was shown to lower the total colony count of environmental microorganisms. However, the critical concentrations of H2O2 detrimental to colony formation on the agar plate remain largely undetermined. Herein, we elucidated the specific effect of H2O2 on microbial colony formation on solid agar medium by external supplementation of varying amounts of H2O2. While common laboratory strains (often called domesticated microbes) formed colonies in the presence of high H2O2 concentrations (48.8 μM or higher), microbes from a freshwater sample demonstrated greatly decreased colony counts in the presence of 8.3 μM H2O2. This implies that environmental microbes are susceptible to much lower concentrations of H2O2 than laboratory strains. Among the emergent colonies on agar plates supplemented with different H2O2 concentrations, the relative abundance of betaproteobacterial colonies was found to be lower on plates containing higher amounts of H2O2. Further, the growth of the representative betaproteobacterial isolates was completely inhibited in the presence of 7.2 μM H2O2. Therefore, our study clearly demonstrates that low micromolar levels of H2O2 in agar plates critically affect growth of environmental microbes, and large portions of those are far more susceptible to the same than laboratory strains. IMPORTANCE It is well-known that most of environmental microorganisms do not form colonies on agar medium despite that agar medium is the commonly used solidified medium. We previously demonstrated the negative effects of H2O2 generation during agar medium preparation on colony formation. In the present study, we investigated the independent effect of H2O2 on microbial growth by adding different concentrations of H2O2 to agar medium. Our results demonstrate for the first time that even low micromolar levels of H2O2 in agar plates, that are far lower than previously recognized as significant, adversely affect colony number obtained from freshwater inoculum. |
| first_indexed | 2024-04-11T12:13:37Z |
| format | Article |
| id | doaj.art-253ffe9a1cb04edfaae43b16779b830a |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-11T12:13:37Z |
| publishDate | 2022-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-253ffe9a1cb04edfaae43b16779b830a2022-12-22T04:24:25ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-12-0110610.1128/spectrum.03336-22Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental StrainsMotoyuki Watanabe0Kensuke Igarashi1Souichiro Kato2Yoichi Kamagata3Wataru Kitagawa4Graduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, JapanGraduate School of Global Food Resources, Hokkaido University, Sapporo, Hokkaido, JapanGraduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, JapanGraduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, JapanGraduate School of Agriculture, Hokkaido University, Sapporo, Hokkaido, JapanABSTRACT We previously showed that autoclaving in preparing agar media is one of the sources of hydrogen peroxide (H2O2) in the medium. This medium-embedded H2O2 was shown to lower the total colony count of environmental microorganisms. However, the critical concentrations of H2O2 detrimental to colony formation on the agar plate remain largely undetermined. Herein, we elucidated the specific effect of H2O2 on microbial colony formation on solid agar medium by external supplementation of varying amounts of H2O2. While common laboratory strains (often called domesticated microbes) formed colonies in the presence of high H2O2 concentrations (48.8 μM or higher), microbes from a freshwater sample demonstrated greatly decreased colony counts in the presence of 8.3 μM H2O2. This implies that environmental microbes are susceptible to much lower concentrations of H2O2 than laboratory strains. Among the emergent colonies on agar plates supplemented with different H2O2 concentrations, the relative abundance of betaproteobacterial colonies was found to be lower on plates containing higher amounts of H2O2. Further, the growth of the representative betaproteobacterial isolates was completely inhibited in the presence of 7.2 μM H2O2. Therefore, our study clearly demonstrates that low micromolar levels of H2O2 in agar plates critically affect growth of environmental microbes, and large portions of those are far more susceptible to the same than laboratory strains. IMPORTANCE It is well-known that most of environmental microorganisms do not form colonies on agar medium despite that agar medium is the commonly used solidified medium. We previously demonstrated the negative effects of H2O2 generation during agar medium preparation on colony formation. In the present study, we investigated the independent effect of H2O2 on microbial growth by adding different concentrations of H2O2 to agar medium. Our results demonstrate for the first time that even low micromolar levels of H2O2 in agar plates, that are far lower than previously recognized as significant, adversely affect colony number obtained from freshwater inoculum.https://journals.asm.org/doi/10.1128/spectrum.03336-22hydrogen peroxideagar plategrowthcolony formationlaboratory strainsenvironmental strains |
| spellingShingle | Motoyuki Watanabe Kensuke Igarashi Souichiro Kato Yoichi Kamagata Wataru Kitagawa Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains Microbiology Spectrum hydrogen peroxide agar plate growth colony formation laboratory strains environmental strains |
| title | Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains |
| title_full | Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains |
| title_fullStr | Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains |
| title_full_unstemmed | Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains |
| title_short | Critical Effect of H2O2 in the Agar Plate on the Growth of Laboratory and Environmental Strains |
| title_sort | critical effect of h2o2 in the agar plate on the growth of laboratory and environmental strains |
| topic | hydrogen peroxide agar plate growth colony formation laboratory strains environmental strains |
| url | https://journals.asm.org/doi/10.1128/spectrum.03336-22 |
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