I wound like to evaluate the biomass of biofilm by using crystal violet. The bacteria was seeded on a porcine skin and cultured for 48 h to form biofilm. I than used crystal violet to stain the biofilm. However, I found the skin tissue it self will also be stained and not able to be washed by water. So I wound like to know if crystal violet can be used for biomass evaluation for biofilm cultured on biological tissues. I only see people evaluate biofilm biomass on 96 -well plate by using crystal violet.
The crystal violet protocol is generally used as a semi-quantitative or qualitative test. It is not possible to use it as you intended in your experiment not only because you are using an animal tissue that will stain too, but also because it is usually used to compare treatments at in vitro testing or to show the capacity of a specific strain or isolate to produce a biofilm. I do not believe the test can be used in an "environmental" application.
the results will be expected since crystal violet as basic dye ... you can avoid using 96 plates and use test tubes and manipulate the procedure to give what you want ... good luck
Hi Fan Zhao ,
As others have noted above, crystal violet will stain the tissue you use as a growth medium, so assessment of the biofilm directly won't be possible.
Your best bet here would be to mechanically remove the biofilm from the tissue following growth. You may need to experiment with different tools (i.e. swab, scalpel, solvent rinse) to find out what works best. I would suggest a simple matrix, like PBS, to do so. Once you harvest your biofilm(s) as a suspension, the approach is up to you, but the following would be my suggestion:
-Gently vortex or disturb the sample to suspend planktonic cells. Quantify them by OD600, or ideally, serial dilution, plating, and colony counting for CFU/mL.
--Remove the supernatant and wash the biofilm in PBS up to three times. It may be convenient to collect the matrix by centrifugation, or simply allow it to settle in the tube depending on how delicate it is.
-Stain in a crystal violet solution for a few mins. Remove and wash well with PBS 3x or until the supernatant runs clear.
-Resolubilize any crystal violet bound to the matrix with the addition of 10% v/v acetic acid. Measure the abs at 590nm and dilute if necessary. If dilution is needed, adjust OD590 by dilution factor so all values measured can be compared.
-Generally, the relative biofilm biomass is calculated as the crystal violet A590nm divided by the OD600 or the culture density in CFU/mL.
This approach would allow you to compare different tissue types and growth conditions of the biofilms in a semi-quantitative way, but certainly has limitations.
Best of luck with your experiment.