As explained by Miral, you can use safranin or other fluorescent dye. However, crystal violet give well results and you can determine either 570 or 590 nm in microplate assays using ELISA reader or microplate reader with monochromator. The main in this, to stain adhered and biofilm forming bacteria after washing non-adherent cells. Any bacterial stain can give result, if you want to compare visually.

Thanks for the answers.

But what will these dyes/stains bind to? The bacterial cells or the extracellular matrix?

Crystal violet and safranin stain bacteria in biofilm layer like Gram staining, whereas, these dyes cannot bind microplate surfaces. Therefore, only bacteria in biofilm layer stained. 

The FilmTracer™ calcein green biofilm stain (Thermo) works as a biofilm stain. The stain is an esterase substrate (Calcein AM) that is non-fluorescent until non-specific esterases hydrolyze it to a green-fluorecent calcein. Other stains include SYPRO Ruby that stains the biofilm matrix. We have used SYTO 9 effectively to study sessile bacteria in complex biofilms. Best of luck!

Thanks a lot everyone

There are several methods which additionaly characterize the biofilm formation. One is the CFU /ml of the biofilm cells. In this method you:

- wash the unattached cells

-add the ringer solution

-use the ultrasonic bath to de-attached cells (not to kill them!)

-dilute and plate the samples

These are described in:

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Hoewer, we just optimise and published one excelent method based on PCR for direct, accurable and specific quantification of attached cells. Here we use dPCR for direct quantification of standard curve and qPCR for ct determination of the sample. Please read:

Article Antiadhesion activity of juniper (Juniperus communis L.) pre...

Enjoy your science!!!

Anja