Methods are provided for the culture of neutrophilic Fe-oxidizing bacteria (FeOB) that grow under microaerobic and anaerobic conditions. For oxygen-requiring lithotrophic Fe oxidizers, it is essential that both O2 and Fe(II) concentrations are kept low, but that an adequate flux of both O2 and Fe(II) are provided to support growth. Techniques using opposing gradients of Fe(II) and O2 are discussed. Preparation of stock solutions of FeS and FeCl2 are described. Methods for the culture of anaerobic FeOB that utilize nitrate or light (anoxygenic photosynthesis) are discussed and presented briefly.