Hi,
I used a protein-based substrate to analyze the biofilm growth. The OD results after Crystal Violet staining of substrate before inoculation showed that crystal violet (CV) attached to the surface in the absence of any biofilm or bacteria, representing high absorbance data.
After inoculation of surfaces in bacterial culture, the OD result was a little bit less than the result for substrates without inoculation.
I would like to know, is there any possibility that crystal violet passes through the biofilm structure, and stains both the surface and biofilm?
Since the OD result for the surface before inoculation (the control one) was higher than the one with biofilm formation, the control subtraction is not a possible way.
I would appreciate if anybody can help me in this regard.
P.S: The protein-based substrates has positive charges.
Crystal violet will bind both protein , DNA and EPS from biofilms. Protein might also bind to the surfaces that you grow your biofilm on . If you use high-bind or ELISA plates then the protein will definitely bind to the surface and the crystal violet will bind to the protein .
Dear Ima
Crystal violet binds to biofilm and passes its layers including protein please read the attached paper
with best regards
Dear Gerry, thank you for your response,
So, the absorbance data is the mixture of crystal violet binds to both biofilm and the protein-based substrate. Therefore, CV is not an adequate method for analysis of biofilm growth on to the protein-based substrates. Isn't it?
Dear Marwa,
Thank you for your response and the attached paper.
So, it seems that having a protein-based material instead of the conventional well plates, interferes the final results of absorbance, as crystal violet not only stains the biofilm, but also passes through the biofilm structure and stains the material beneath.
Dear Ima,
I think that you have got good answers to your question about using Crystal violet to measure biofilm-formation on solid surfaces. However, you could use any number of stains if you wanted to measure bacterial attachment and biomass, and one or other of these might not bind so strongly to the prepared surfaces. You could consider using a DNA stain (and this might also detect eDNA), or a metabolic substrate that would stir live cells. Alternatively you could measure directly live cells by plating them out.
Your measurements of the before and after-inoculation suggests that the first colonising cells may be removing or degrading some of the Crystal violet on the support, or that the washing procedure removes more material when bacterial cells are attached. You may be able to see a bigger difference if you incubated the cultures for longer to produce larger biofilms, and you could consider using a higher concentration of Crystal violet to saturate the substrate first.
Regards, Andrew.
Dear Dr. Andrew,
Many thanks for your helpful response.
Your suggestions about other methods rather than Crystal Violet are so beneficial and I will use them in my future work.
The substrate that I used was collagen that is swelling in the aqueous media. You are completely true. If I incubate the samples for longer time, probably I can get better results, since larger biofilm may reduce the Crystal Violet penetration to the substrate structure.
However, what do you mean as using a higher concentration to saturate the substrate first? With the high OD results for Crystal Violet staining of collagen, it seems that Crystal Violet adsorbs easily to the collagen and de-colorizing using ethanol can easily remove it from the substrate. I think the decolorizing agent may even be more influential on collagen than the lipid structure of bacterial cells for removing the Crystal Violet. In this case, is higher concentration of Crystal Violet (for substarte saturation), helpful for the biofilm detection?
Thank you again,
Ima
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