Ten µl (if tested with TA98) or 40 µl (if tested with TA100) of 1-aminopyrene (0.0375 mg/ml) was added into a tube, followed by 710-740 µl of 0.2 N hydrochloric acid (sufficient to acidify the reaction mixture to pH 3.0 - 3.4) and 250 µl of 2M sodium nitrite with thorough mixing.