Critical Effect of HO 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 (HO) in the medium. This medium-embedded HO was shown to lower the total colony count of environmental microorganisms. However, the critical concentrations of HO detrimental to colony formation on the agar plate remain largely undetermined. Herein, we elucidated the specific effect of HO on microbial colony formation on solid agar medium by external supplementation of varying amounts of HO. While common laboratory strains (often called domesticated microbes) formed colonies in the presence of high HO concentrations (48.8 μM or higher), microbes from a freshwater sample demonstrated greatly decreased colony counts in the presence of 8.3 μM HO. This implies that environmental microbes are susceptible to much lower concentrations of HO than laboratory strains. Among the emergent colonies on agar plates supplemented with different HO concentrations, the relative abundance of betaproteobacterial colonies was found to be lower on plates containing higher amounts of HO. Further, the growth of the representative betaproteobacterial isolates was completely inhibited in the presence of 7.2 μM HO. Therefore, our study clearly demonstrates that low micromolar levels of HO in agar plates critically affect growth of environmental microbes, and large portions of those are far more susceptible to the same than laboratory strains. 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 HO generation during agar medium preparation on colony formation. In the present study, we investigated the independent effect of HO on microbial growth by adding different concentrations of HO to agar medium. Our results demonstrate for the first time that even low micromolar levels of HO in agar plates, that are far lower than previously recognized as significant, adversely affect colony number obtained from freshwater inoculum.