Biological indicators may be improperly activated. The disclosed subject matter is directed to methods of confirming that a biological indicator having an ampule containing a growth medium has been properly activated such that it may be assayed. The methods may include the steps of measuring a first fluorescence intensity of the biological indicator, heating the biological indicator； quenching the fluorescence intensity of the biological indicator from the first fluorescence intensity to a second fluorescence intensity, measuring the second fluorescence intensity； comparing the second fluorescence intensity and first fluorescence intensity to obtain a comparison value； and determining that the comparison value corresponds to a quenching metric of the liquid growth medium.
