3 replies to this topic

[ales]

Hi!

I'm trying to use the crystal violet assay to determine cell viability but I'm experiencing some technical difficulties… I can’t completely extract the crystal violet dye from the cells and I still have some “violet” on the bottom of the wells. The weird thing is that one week ago I had no problems. The solutions are not the same than one week ago but the batches of all my components are not changed.

Protocol:
- Remove the media
- Wash the plates with PBS 1x (2 times)
- Add 50 ul crystal violet staining solution:
   - crystal violet 0.5 %  (m/v)
   - MetOH 25 %
   - H2O
- Incubate 5’ at RT
- Wash the plate
- Dry the plate
- Add 250 µL of the following solution:

   - Sodium citrate 0.1 M

         - EtOH 50 %

   - H2O

- Incubate 30’ at RT
- Read the absorbance at 570 nm

Obviously, I tested different solutions to extract the dye (like 33% acetic acid, DMSO, 0.1% SDS) but I could not solve my problem. Can someone help me? I’m afraid that I will not be able to read all my plates that are already stained.

Thanks,
Ale

[bob1]

The crystal violet stains proteins, you probably have protein residues on the wells.  I don't think you will be able to overcome this.

Also note that crystal violet will precipitate with storage - you could re-filter and try again.

[ales]

Hi,
I prepared one new solution of crystal violet and I had the same problem. Before I was able to  extract completely the crystal violet from my cells and now no. I can't understand....

[Adrian K]

I used to work with and use crystal violet to stain biofilm. However, after staining with CV, I use 100% methanol to elute it. No issues.