Hello! We are quantifying by crystal violet the biofilm formation of some isolates. However, we have experienced some problems such as, at the time of removing the excess crystal violet, we always have too much left in our controls (media without bacteria). We have thought of performing washes with 10% ethanol, however, we have not found any literature that tells us if it is valid to use it or if there are other ways to remove as much crystal violet as possible.
We appreciate your contributions and suggestions.
Thank you!
I think it largely depends on the materials and methodology you are using. Any answer would be mere guesswork without more details.
Hello,
We are working in 96 well plates, leaving to incubate with crystal violet for 15 min and then making washes with water (as many as necessary), and when we see that the water comes out clear, we proceed to make an incubation for 15 min with absolute ethanol, to recover the crystal violet of the biofilms, but that's when we also get coloration in the blanks.
I do not know if this explanation helps. Thank you.
96 well plates are usually made of polystyrene, which is very easily corroded by organic solvents. If the crystal violet is dissolved in ethanol or some other organic solvent, then it could penetrate into the polystyrene. I used a 0.4% aqueous solution of crystal violet for 15min, then washed the wells 3x with PBS and the bioflm bound crystal violet was solubilized in each well by the addition of 200 μl 33% acetic acid. I don't remember exactly, but there always was a slight coloration in the negative wells. I guess it's just the nature of the polystyrene polymer. Maybe you could make a small test in glass or polypropylene tubes (eppendorf, pcr tubes) to verify your method.
Thank you very much for your help.
We gonna check in other containers as you said.
thanks!!!
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