Biofilm amount estimation by fluorescein diacetate

Various methods for direct and indirect biofilm amount estimation are available but most of them have been developed on free cell cultures and/or their application to biofilm analysis often implies biofilm removal from the solid surface or extraction procedures. This work presents a method to measure biofilm bacterial activity, that does not require the removal step. It uses fluorescein diacetate (FDA), a fluorogen (fluorescein) conjugated to two acetate radicals. This compound, once hydrolyzed by an exoenzyme that is present in almost all bacteria, releases the fluorogen that has an absorbance at 490 nm. Experimental tests have shown a good correlation between the absorbance of the solution at 490 nm and bacterial concentrations. This method may be particularly helpful in potable water pipe control to indicate where and when it is necessary to perform corrective actions to stop and/or reduce biofilm growth.