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The classical approach for the cultivation of neutrophilic microaerophilic Fe(II)-oxidizing bacteria is agar-based gradient tubes where these bacteria find optimal growth conditions in opposing gradients of oxygen (O2) and dissolved Fe(II) (Fe2+). The goals of this study were to quantify the temporal development of O2 and Fe2+ concentrations over time, to compare abiotic and microbially inoculated tubes and to test the suitability of different Fe(II)-sources for the cultivation of freshwater and marine microaerophilic Fe(II)-oxidizers. O2 and Fe2+ gradients were monitored on a high spatial resolution as a function of time applying amperometric and voltammetric microsensors. Fe(II)-oxidizers could be cultivated well with FeS and zero-valent iron powder as Fe(II)-source, but FeCO3 and FeCl2 are extremely sensitive for this application. Fe(III) minerals accumulated in inoculated tubes within the first days in regions with an O2 concentration of 20-40 μM and were confirmed to be related to bacterial growth. Microbial Fe(II) oxidation could compete only for the first days with the abiotic reaction after which heterogeneous Fe(II) oxidation, catalyzed by Fe(III) minerals, dominated. Our results imply that transfer of cultures to fresh tubes within 48-72 h is crucial to provide optimal growth conditions for microaerophilic Fe(II)-oxidizers, particularly for the isolation of new strains.