In this paper, the development of a novel means of investigating the attachment and subsequent biofilm formation of mineral bioleaching micro-organisms to mineral surfaces in situ is described. The protocol was developed to investigate the interactions of micro-organisms with sulfide minerals and low-grade chalcopyrite ore under conditions resemblant of a bioheap environment. The method makes use of a biofilm reactor in which thin sections of mineral ore are mounted. The reactor is operated as a continuous flow-through system. Attachment of pure and mixed cultures of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum is assessed. The technique allows for the investigation of microbial ecology with special regard to microbe-mineral attachment, site and mineral specific associations of micro-organisms and spatial organisation of microbial communities present through the use of fluorescent microscopy techniques. Preliminary fluorescent in situ hybridisation (FISH) analysis of the attachment of L ferriphilum and A. ferrooxidans to massive chalcopyrite sections, as well as to low-grade chalcopyrite containing ore sections is presented. In the case of both low-grade and massive sulfide mineral samples, attachment of mixed micro-colonies was observed in regions where surface defects were prevalent. In low-grade samples, preferential attachment was observed in regions where sulfide minerals were present. The density of the attached micro-colonies increased with an increase in contacting time (from 20, 72 and 96 h) and was indicative of an actively growing mono-layered biofilm. (C) 2010 Published by Elsevier Ltd.