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Acidiphilium cryptum is a heterotrophic acidophile commonly found in acidic environments and industrial bioleaching operations. Attachment to mineral surfaces may serve to maintain this organism in aqueous environments where it is subject to removal by hydrodynamic forces. Using indirect and direct methods we have looked at the binding of A.cryptum to chalcopyrite (CuFeS₂) and other mineral ores to determine whether specific adhesins mediate binding. A modified ELISA binding assay (the Ore ELISA) was developed to measure direct adherence. Finely ground chalcopyrite was bound irreversibly to the walls of an ELISA plate, the organisms were added and after incubation and washing, the number of attached bacteria were assessed by reacting with anti-A.cryptum antibody followed by goat anti-rabbit IgG conjugated to alkaline phosphatase. This assay was found to be sensitive, rapid and reproducible. The Ore ELISA allowed direct binding measurement in the presence of various inhibitors and provided a rapid screening method for adherence-defective mutants. Adherence was shown to be saturable and increased slightly as pH decreased. A moderate increase in binding affinity was recorded in the presence of monovalent and divalent cations and EDTA. Various bactericidal agents and pentose and hexose sugars had no effect on chalcopyrite attachment. Reducing agents had little effect on cell adherence. A strong increase in adherence was observed in the presence of surface active agents. Bovine serum albumin and gelatin were both found to markedly reduce mineral surface binding. Competition for attachment sites between A.cryptum and the autotrophic acidophile, Thiobacillus ferrooxidans, showed that each organism binds to unique sites on the chalcopyrite surface. A.cryptum mutant strains displaying reduced adherence to chalcopyrite were shown to lack a 31.6 kDa outer membrane protein. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate