Dear Colleagues

We are using a protocol to assess amount of biofilm produced by Acinetobacter. Basically biofilms are first fixed with 99% methanol and then stained with crystal violet (from Gram staining kit) for 15 min. The stain is decanted and washed with Phosphate buffered saline pH 7.4 for thrice (We even tried washings with water). The films are then overlaid with Glacial Acetic Acid and Optical density measured at 630nm wavelength.

Often our sample (Acinetobacter treated with inhibitory chemicals)OD is lower than the blank (well without bacteria but treated similarly for staining with crystal violet protocol mentioned above).

OD is measured with ELISA reader.

We don't make a homogenous suspension in glacial acetic acid -I suspect if this is right to do (?)

any expert opinion would be welcome.

Do we miss out something that's crucial for estimating biofilm (indirectly) with crystal violet staining? Why our OD for Acinetobacter treated with inhibitory chemicals is lesser than the blank?