Critical Effect of H₂O₂ in the Agar Plate on the Growth of Laboratory and Environmental Strains

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.