This study provides a technologically feasible strategy from a practical perspective for in-situ isolation of stable Thiobacillus thiooxidans. Quantitative assessment also clearly emphasizes the evolutionary outcome of enrichment appearing in dichotomous guise (i.e., success or failure; "yes/no"). Progressive increasing in acid formation capability presents the promising viability of selection scheme and stability of T. thiooxidans isolates. Incorporation of polyene antibiotics-nystatin and exclusion of solid particles efficiently evacuates possible contaminant species from soil-dilution samples to guarantee an ecologically stable T. thiooxidans to be resulted. Asymptotically convergent values of sulfuric acid formation rate strongly support high purity of ecologically enriched T. thiooxidans isolates. Further evidence in 16S rDNA detection clearly confirmed pure acidophilic isolate as T. thiooxidans, promising feasibility of our proposed scheme for serial isolation. This selection scheme provides a theoretically plausible and economically appropriate strategy to screen acidophilic T. thiooxidans for practical in situ applications.