A biological indicator is formed from two members (or a sheet with one portion folded onto another portion) which are sealed together and form a pathway having three sections. One section opens to the exterior and is preferably configured as a tortuous path. A middle pathway portion downstream of the tortuous path houses a frangible ampule. The frangible ampule contains growth medium for microorganisms. An interior pathway portion (most downstream from the exterior) houses a carrier inoculated with spores of a microorganism. The biological indicator preferably is prepared by a process in which a thermoplastic sheet has the pathway formed by thermoforming and the sealing is by heat sealing.