Bioleaching is the dissolution of metal sulfides, such as pyrite and chalcopyrite, by bacterial oxidation processes. It has been found that attachment of leaching bacteria to the mineral surface enhances the metal sulfide dissolution. The interaction of mixed cultures with respect to initial attachment processes has not been investigated. Therefore in this study we quantified and visualized initial colonization on pyrite by pure and mixed cultures. Strains of the genera Acidithiobacillus and Leptospirillum were tested. Sessile and planktonic cells were visualized by fluorescence microscopy using DAPI. FISH, Syto (TM) 9, lectin- and calcofluor-staining. Additionally, atomic force microscopy (AFM) was used for the investigations on cell morphology, spatial arrangement of cells on pyrite and mineral surface topography. The morphology of planktonic and sessile cells is different. Moreover, planktonic cells show differences in morphology due to the use of different substrata. By using different visualization methods it could be proven that colonization and biofilm formation on pyrite in mixed cultures is mostly dominated by Leptospirillum spp. Interactions of different species resulted in increased production of extracellular polymeric substances (EPS) or caused bacteria showing little tendency to attach when in monoculture to be incorporated into a biofilm by those that attach preferentially. Consequently, biofilm formation and metabolic diversity were furthered. One of the most important results is the finding that not all bioleaching bacteria are involved to the same extent in biofilm formation. Thus, further work shall allow us elucidate the important bacteria for biotechnological use, thereby leaching processes can be faster, more efficient and costs can be reduced. (C) 2010 Elsevier Ltd. All rights reserved.