Precise quantification of biofilm is critical as the formation and persistence of biofilm have significant implications in the environmental, therapeutic and industrial contexts. The microtiter plate assay using crystal violet with 33% glacial acetic acid or 94-100% ethanol as the resolubilising agent is widely used for the categorisation of biofilms into weak, moderate and strong categories. But, the use of varying wavelengths for the measurement of biofilm resulted in discrepancies in categorisation across the studies due to the difference in the extinction coefficient of CV. This study emphasises the importance of measuring the biofilm at the absorbance maximum (λ