Biofilms formed by biofilm forming ATCC strains in 96- non culture & culture treated well plates and fixed with methanol tend to partially dislodge during washing and dye dissolution steps. One species form too thick biofilms when stained and was unable to be read with microplate reader, hence ethanol:acetone solvent had be transferred to new wells and read of their OD. These 2 issues may lead to faulty quantification of biofilms. To avoid these errors, can biofilm cut-off values and formation index be calculated with biofilm formed in wells removed of its broth, dried, fixed with methanol, dried and fresh broth added just before reading (with negative control being broth without organisms)? Will this be a good estimation of formed biofilms?
However, then again it comes to how do we differentiate between a thick or thin biofilm formation without the staining...
Dear Ramona
When using the crystal violet technique, the same procedure must be applied to all the wells,including the controls. A simple protocol would be fixation with ethanol, staining with CV and acetic acid for the dissolution and ressuspension of the fixed mass. You may dilute prior to reading if the OD is to high. I used thit method in my work:
http://www.sciencedirect.com/science/article/pii/S0964830513002242.
Dear Madalena... I actually optimized the protocol steps later and found that all the steps you have mentioned (fixing with alcohol - i used methanol), staining with CV, acetic acid for dissolution and resuspension and diluting the acid solution) was found to be effective for my assays too.. thanks a lot!
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